Stanley Dean Rider

PICKLE Acts throughout the Plant to Repress Expression of Embryonic Traits and May Play a Role in Gibberellin-Dependent Responses

A seed marks the transition between two developmental states; a plant is an embryo during seed formation, whereas it is a seedling after emergence from the seed. Two factors have been identified in Arabidopsis that play a role in establishment of repression of the embryonic state: PKL (PICKLE), which codes for a putative CHD3 chromatin remodeling factor, and gibberellin (GA), a plant growth regulator. Previous observations have also suggested that PKL mediates some aspects of GA responsiveness in the adult plant. To investigate possible mechanisms by which PKL and GA might act to repress the embryonic state, we further characterized the ability of PKL and GA to repress embryonic traits and reexamined the role of PKL in mediating GA-dependent responses. We found that PKL acts throughout the seedling to repress expression of embryonic traits. Although the ability of pkl seedlings to express embryonic traits is strongly induced by inhibiting GA biosynthesis, it is only marginally responsive to abscisic acid and SPY (SPINDLY), factors that have previously been demonstrated to inhibit GA-dependent responses during germination. We also observed that pkl plants exhibit the phenotypic hallmarks of a mutation in a positive regulator of a GA response pathway including reduced GA responsiveness and increased synthesis of bioactive GAs. These observations indicate that PKL may mediate a subset of GA-dependent responses during shoot development.

The CHD3 Remodeler PICKLE Promotes Trimethylation of Histone H3 Lysine 27

CHD3 proteins are ATP-dependent chromatin remodelers that contribute to repression of developmentally regulated genes in both animal and plant systems. In animals, this repression has been linked to a multiple subunit complex, Mi-2/NuRD, whose constituents include a CHD3 protein, a histone deacetylase, and a methyl-CpG-binding domain protein. In Arabidopsis, PICKLE (PKL) codes for a CHD3 protein that acts during germination to repress expression of seed-associated genes. Repression of seed-associated traits is promoted in pkl seedlings by the plant growth regulator gibberellin (GA). We undertook a microarray analysis to determine how PKL and GA act to promote the transition from seed to seedling. We found that PKL and GA act in separate pathways to repress expression of seed-specific genes. Comparison of genomic datasets revealed that PKL-dependent genes are enriched for trimethylation of histone H3 lysine 27 (H3K27me3), a repressive epigenetic mark. Chromatin immunoprecipitation studies demonstrate that PKL promotes H3K27me3 in both germinating seedlings and in adult plants but do not identify a connection between PKL-dependent expression and acetylation levels. Taken together, our analyses illuminate a new pathway by which CHD3 remodelers contribute to repression in eukaryotes.

Metabolic profiling of the Arabidopsis pkl mutant reveals selective derepression of embryonic traits

Embryos express several unique differentiation characteristics, including the accumulation of a number of metabolites that are generally considered to be unique to seeds. PICKLE (PKL) codes for a CHD3-chromatin remodeling factor that is necessary for repression of embryonic traits in seedlings of Arabidopsis thaliana (L.) Heynh. In pkl mutants, primary roots are capable of expressing many embryonic traits after germination and are referred to as “pickle roots”. In an attempt to examine the breadth of PKL-dependent repression of embryo-specific differentiation pathways, we determined the extent to which a variety of embryo-specific compounds accumulate in pickle roots. We found that pickle roots accumulate triacylglycerol with a fatty acid composition that is similar to that found in seeds. The major seed storage proteins are also present in pickle roots. In addition to these two well-characterized seed storage compounds, we observed that pickle roots accumulate phytate, a form of stored phosphate that is preferentially accumulated in seeds. Seeds of members of the Brassicaceae also accumulate a variety of unique secondary metabolites, including sinapate esters and glucosinolates. Surprisingly, the levels of secondary metabolites in pickle roots were not suggestive of an embryonic differentiation state, but did reveal that a mutation in PKL results in substantial changes in root secondary metabolism. Taken together, these data suggest that PKL is responsible for regulating some but not all aspects of the embryonic program as it relates to the accumulation of embryo-specific metabolites.

Cryptosporidium: genomic and biochemical features.

Recent progress in understanding the unique biochemistry of the two closely related human enteric pathogens Cryptosporidium parvum and Cryptosporidium hominis has been stimulated by the elucidation of the complete genome sequences for both pathogens. Much of the work that has occurred since that time has been focused on understanding the metabolic pathways encoded by the genome in hopes of providing increased understanding of the parasite biology, and in the identification of novel targets for pharmacological interventions. However, despite identifying the genes encoding enzymes that participate in many of the major metabolic pathways, only a hand full of proteins have actually been the subjects of detailed scrutiny. Thus, much of the biochemistry of these parasites remains a true mystery.

Chromatin-remodelling proteins of the pea aphid, Acyrthosiphon pisum (Harris)

Aphids display extraordinary developmental plasticity in response to environmental cues. These differential responses to environmental changes may be due in part to changes in gene expression patterns. To understand the molecular basis for aphid developmental plasticity, we attempted to identify the chromatin‐remodelling machinery in the recently sequenced pea aphid genome. We find that the pea aphid possesses a complement of metazoan histone modifying enzymes with greater gene family diversity than that seen in a number of other arthropods. Several genes appear to have undergone recent duplication and divergence, potentially enabling greater combinatorial diversity among the chromatin‐remodelling complexes. The abundant aphid chromatin modifying enzymes may facilitate the phenotypic plasticity necessary to maintain the complex life cycle of the aphid.

Genetics of esterase-mediated insecticide resistance in the aphid Schizaphis graminum

The genetics of organophosphate resistance, including the pattern of inheritance and the underlying genetic mechanism, were investigated in the aphid Schizaphis graminum (the greenbug). Resistant greenbugs with pattern 1 (R1) and pattern 2 (R2) esterase and susceptible (S) greenbugs were induced into the sexual cycle and crossed. Each resistance-associated esterase pattern in the greenbug was inherited in a Mendelian fashion as a single gene. An aphid clone that displayed both resistance-associated esterase patterns was produced in the F1 generation of a cross between R2 and R1 insects, and was designated pattern 3 (R3). DNA from different R1 aphids and the R3 aphid clone displayed a restriction fragment pattern different from that of R2 and S clones, and about a twofold increase in hybridization compared with S aphids. DNA from R2 clones showed no restriction fragment differences or increased hybridization signal compared to S insects. These findings suggest that the mechanism behind the R1 esterase activity may be gene amplification, but that the R2 elevated esterase activity may result from a different genetic mechanism.

Differential expression of the two distinct replication protein A subunits from Cryptosporidium parvum

Apicomplexan parasites differ from their host by possessing at least two distinct types (long and short) of replication protein A large subunits (RPA1). Different roles for the long and short types of RPA1 proteins have been implied in early biochemical studies, but certain details remained to be elucidated. In the present study, we have found that the Cryptosporidium parvum short‐type RPA1 (CpRPA1A) was highly expressed at S‐phase in parasites during the early stage of merogony (a cell multiplication process unique to this group of parasites), but otherwise present in the cytosol at a much lower level in other cell‐cycle stages. This observation indicates that CpRPA1A is probably responsible for the general DNA replication of the parasite. On the other hand, the long‐type CpRPA1B protein was present in a much lower level in the early life cycle stages, but elevated at later stages involved in sexual development, indicating that CpRPA1B may play a role in DNA recombination. Additionally, CpRPA1B could be up‐regulated by UV exposure, indicating that this long‐type RPA1 is probably involved in DNA repair. Collectively, our data implies that the two RPA1 proteins in C. parvum are performing different roles during DNA replication, repair and recombination in this parasite. J. Cell. Biochem. 104: 2207–2216, 2008.

A mixture model approach for the analysis of small exploratory microarray experiments

The microarray is an important and powerful tool for prescreening of genes for further research. However, alternative solutions are needed to increase power in small microarray experiments. Use of traditional parametric and even non-parametric tests for such small experiments lack power and have distributional problems. A mixture model is described that is performed directly on expression differences assuming that genes in alternative treatments are expressed or not in all combinations (i) not expressed in either condition, (ii) expressed only under the first condition, (iii) expressed only under the second condition, and (iv) expressed under both conditions, giving rise to 4 possible clusters with two treatments. The approach is termed a Mean-Difference-Mixture-Model (MD-MM) method. Accuracy and power of the MD-MM was compared to other commonly used methods, using both simulations, microarray data, and quantitative real time PCR (qRT-PCR). The MD-MM was found to be generally superior to other methods in most situations. The advantage was greatest in situations where there were few replicates, poor signal to noise ratios, or non-homogenous variances.

Mutations in Liver X Receptor Alpha that Impair Dimerization and Ligand Dependent Transactivation

Liver X receptor alpha (LXRα) is crucial for the maintenance of lipid and cholesterol homeostasis. Ligand binding and dimerization with retinoid X receptor (RXR) or peroxisome proliferator-activated receptor (PPAR) is required for forming active DNA binding complexes leading to gene regulation. Structure based prediction and solvent accessibility of LXRα LBD shows that residues H383, E387, H390, L414, and R415 which are located in helices 9 and 10 may be critical for mediating protein-protein interactions. In this study, LXRα interface residues were individually mutated to determine their effects on ligand binding, protein-protein association, subcellular localization, and transactivation activity. LXRα L414R and R415A lacked binding to T-0901317, but retained binding to 25-Hydroxycholesterol. In vitro assay and a cell based assay demonstrated that LXRα L414R was specifically impaired for interactions with RXRα but not PPARα suggesting that charge reversal at the interface provides selectivity to LXRα dimerization. Furthermore, binding of LXRα L414R or R415A with PPARα exhibited minimal conformational changes in the dimer secondary structure. Interestingly, all LXRα mutants exhibited lower levels of ligand dependent luciferase activity driven by the SREBP-1c or ApoA1 promoter. Taken together, our data demonstrates that intact hydrophobic interactions and salt bridges at the interface mediate efficient ligand-dependent transactivation activities.

The complete mitochondrial genome of the desert darkling beetle Asbolus verrucosus (Coleoptera, Tenebrionidae)

Abstract The complete mitochondrial genome of the desert darkling beetle Asbolus verrucosus (LeConte, 1851) was sequenced using paired-end technology to an average depth of 42,111× and assembled using De Bruijn graph-based methods. The genome is 15,828 bp in length and conforms to the basal arthropod mitochondrial gene composition with the same gene orders and orientations as other darkling beetle mitochondria. This arrangement includes a control region, 22 tRNA genes, 2 rRNA genes and 13 protein-coding genes. The main coding strand is probably replicated as the lagging strand (GC skew of −0.36 and AT skew of +0.19). Phylogenomics analyses are consistent with taxonomic classifications and indicate that Tenebrio molitor is the closest relative that has a completely sequenced mitochondrial genome available for analysis. This is the first fully assembled mitogenome sequence for a darkling beetle in the subfamily Pimeliinae and will be useful for population studies on members of this ecologically important group of beetles.