Activation domains for controlling plant gene expression using designed transcription factors.

Targeted gene regulation via designed transcription factors has great potential for precise phenotypic modification and acceleration of novel crop trait development. To this end, designed transcriptional activators have been constructed by fusing transcriptional activation domains to DNA-binding proteins. In this study, a transcriptional activator from the herpes simplex virus, VP16, was used to identify plant regulatory proteins. Transcriptional activation domains were identified from each protein and fused with zinc finger DNA-binding proteins (ZFPs) to generate designed transcriptional activators. In addition, specific sequences within each transcriptional activation domain were modified to mimic the VP16 contact motif that interacts directly with RNA polymerase II core transcription factors. To evaluate these designed transcriptional activators, test systems were built in yeast and tobacco comprising reporter genes driven by promoters containing ZFP-binding sites upstream of the transcriptional start site. In yeast, transcriptional domains from the plant proteins ERF2 and PTI4 activated MEL1 reporter gene expression to levels similar to VP16 and the modified sequences displayed even greater levels of activation. Following stable transformation of the tobacco reporter system with transcriptional activators derived from ERF2, GUS reporter gene transcript accumulation was equal to or greater than those derived from VP16. Moreover, a modified ERF2 domain displayed significantly enhanced transcriptional activation compared with VP16 and with the unmodified ERF2 sequence. These results demonstrate that plant sequences capable of facilitating transcriptional activation can be found and, when fused to DNA-binding proteins, can enhance gene expression.

The CaaX specificities of Arabidopsis protein prenyltransferases explain era1 and ggb phenotypes.

BACKGROUND: Protein prenylation is a common post-translational modification in metazoans, protozoans, fungi, and plants. This modification, which mediates protein-membrane and protein-protein interactions, is characterized by the covalent attachment of a fifteen-carbon farnesyl or twenty-carbon geranylgeranyl group to the cysteine residue of a carboxyl terminal CaaX motif. In Arabidopsis, era1 mutants lacking protein farnesyltransferase exhibit enlarged meristems, supernumerary floral organs, an enhanced response to abscisic acid (ABA), and drought tolerance. In contrast, ggb mutants lacking protein geranylgeranyltransferase type 1 exhibit subtle changes in ABA and auxin responsiveness, but develop normally. RESULTS: We have expressed recombinant Arabidopsis protein farnesyltransferase (PFT) and protein geranylgeranyltransferase type 1 (PGGT1) in E. coli and characterized purified enzymes with respect to kinetic constants and substrate specificities. Our results indicate that, whereas PFT exhibits little specificity for the terminal amino acid of the CaaX motif, PGGT1 exclusively prenylates CaaX proteins with a leucine in the terminal position. Moreover, we found that different substrates exhibit similar K(m) but different k(cat) values in the presence of PFT and PGGT1, indicating that substrate specificities are determined primarily by reactivity rather than binding affinity. CONCLUSIONS: The data presented here potentially explain the relatively strong phenotype of era1 mutants and weak phenotype of ggb mutants. Specifically, the substrate specificities of PFT and PGGT1 suggest that PFT can compensate for loss of PGGT1 in ggb mutants more effectively than PGGT1 can compensate for loss of PFT in era1 mutants. Moreover, our results indicate that PFT and PGGT1 substrate specificities are primarily due to differences in catalysis, rather than differences in substrate binding.

Farnesylcysteine lyase is involved in negative regulation of abscisic acid signaling in Arabidopsis.

The Arabidopsis FCLY gene encodes a specific farnesylcysteine (FC) lyase, which is responsible for the oxidative metabolism of FC to farnesal and cysteine. In addition, fcly mutants with quantitative decreases in FC lyase activity exhibit an enhanced response to ABA. However, the enzymological properties of the FCLY-encoded enzyme and its precise role in ABA signaling remain unclear. Here, we show that recombinant Arabidopsis FC lyase expressed in insect cells exhibits high selectivity for FC as a substrate and requires FAD and molecular oxygen for activity. Arabidopsis FC lyase is also shown to undergo post-translational N-glycosylation. FC, which is a competitive inhibitor of isoprenylcysteine methyltransferase (ICMT), accumulates in fcly mutants. Moreover, the enhanced response of fcly mutants to ABA is reversed by ICMT overexpression. These observations support the hypothesis that the ABA hypersensitive phenotype of fcly plants is the result of FC accumulation and inhibition of ICMT.

Protein isoprenylation: the fat of the matter.

Protein isoprenylation refers to the covalent attachment of a 15-carbon farnesyl or 20-carbon geranylgeranyl moiety to a cysteine residue at or near the carboxyl terminus. This post-translational lipid modification, which mediates protein-membrane and protein-protein interactions, is necessary for normal control of abscisic acid and auxin signaling, meristem development, and other fundamental processes. Recent studies have also revealed roles for protein isoprenylation in cytokinin biosynthesis and innate immunity. Most isoprenylated proteins are further modified by carboxyl terminal proteolysis and methylation and, collectively, these modifications are necessary for the targeting and function of isoprenylated proteins.

A discrete-time survival analysis of the relationship between truancy and the onset of marijuana use.

OBJECTIVE: Despite truancy being a common behavior among teenagers, little research has assessed its deleterious effects. In this study, the effect of truancy on the initiation of marijuana use was examined. METHOD: Using data from the Rochester Youth Development Study (a longitudinal sample of predominantly minority youth), discrete time survival analyses were estimated to assess the effect of truancy on the subsequent initiation of marijuana use. The current analyses used 5 years of panel data collected from youth and their primary caregiver every 6 months throughout adolescence. RESULTS: Truancy was a significant predictor of the initiation of marijuana use during each subsequent 6-month period. The effect was more robust in earlier compared with later adolescence. These effects persisted after controlling for potential risk factors that are shared by both truancy and drug use, including commitment to school, grade-point average, delinquent values, prior involvement in delinquency, peer reactions to delinquency, parental monitoring, affective ties to the child, and positive parenting. CONCLUSIONS: We argue that the effect is, in part, the result of reduced social control (i.e., disengagement from pro-social entities such as school) and, in part, the result of the unsupervised, unmonitored time afforded by truancy. Prevention initiatives aimed at reducing truancy also may have a beneficial impact on preventing the initiation of drug use among adolescents.

Isoprenylcysteine methylation and demethylation regulate abscisic acid signaling in Arabidopsis.

Isoprenylated proteins bear an isoprenylcysteine methyl ester at the C terminus. Although isoprenylated proteins have been implicated in meristem development and negative regulation of abscisic acid (ABA) signaling, the functional role of the terminal methyl group has not been described. Here, we show that transgenic Arabidopsis thaliana plants overproducing isoprenylcysteine methyltransferase (ICMT) exhibit ABA insensitivity in stomatal closure and seed germination assays, establishing ICMT as a negative regulator of ABA signaling. By contrast, transgenic plants overproducing isoprenylcysteine methylesterase (ICME) exhibit ABA hypersensitivity in stomatal closure and seed germination assays. Thus, ICME is a positive regulator of ABA signaling. To test the hypothesis that ABA signaling is under feedback regulation at the level of isoprenylcysteine methylation, we examined the effect of ABA on ICMT and ICME gene expression. Interestingly, ABA induces ICME gene expression, establishing a positive feedback loop whereby ABA promotes ABA responsiveness of plant cells via induction of ICME expression, which presumably results in the demethylation and inactivation of isoprenylated negative regulators of ABA signaling. These results suggest strategies for metabolic engineering of crop species for drought tolerance by targeted alterations in isoprenylcysteine methylation.

School-related risk and protective factors associated with truancy among urban youth placed at risk.

Truancy is a serious concern in the United States. Its negative effects are so pervasive that in 2003 the Office of Juvenile Justice and Delinquency Prevention named truancy prevention a national priority. Effective prevention of truancy requires a thorough understanding of the characteristics that describe truant youth as well as factors that may put them at risk for truancy. Unfortunately, surprisingly little is known about the correlates and/or causes of truancy. In this paper we explore associations between truancy and several salient school-related risk and protective factors among a sample of youth who grew up in socially disorganized neighborhoods of Denver, CO. We demonstrate that several school-related risk and protective factors are associated with truancy. Perhaps most importantly, we identify that the two most robust predictors are school performance and involvement with delinquent peers, and that these two variables form a synergistic relationship in which the relationship between delinquent peer association and truancy is mitigated among students who perform well in school. EDITORS' STRATEGIC IMPLICATIONS: The authors use data from a large probability sample drawn from neighborhoods with high crime rates to identify key correlates of truancy. They also draw attention to the dearth of efficacious truancy prevention efforts in spite of the magnitude of the problem.

Arabidopsis thaliana plants possess a specific farnesylcysteine lyase that is involved in detoxification and recycling of farnesylcysteine.

In plants, prenylated proteins are involved in actin organization, calcium-mediated signal transduction, and many other biological processes. Arabidopsis thaliana mutants lacking functional protein prenyltransferase genes have also revealed roles for prenylated proteins in phytohormone signaling and meristem development. However, to date, the turnover of prenylated plant proteins and the fate of the prenylcysteine (PC) residue have not been described. We have detected an enzyme activity in Arabidopsis plants that metabolizes farnesylcysteine (FC) to farnesal, which is subsequently reduced to farnesol. Unlike its mammalian ortholog, Arabidopsis FC lyase exhibits specificity for FC over geranylgeranylcysteine (GGC), and recognizes N-acetyl-FC (AFC). FC lyase is encoded by a gene on chromosome 5 of the Arabidopsis genome (FCLY, At5g63910) and is ubiquitously expressed in Arabidopsis tissues and organs. Furthermore, T-DNA insertions into the FCLY gene cause significant decreases in FC lyase activity and an enhanced response to abscisic acid (ABA) in seed germination assays. The effects of FCLY mutations on ABA sensitivity are even greater in the presence of exogenous FC. These data suggest that plants possess a specific FC detoxification and recycling pathway.

Truancy's effect on the onset of drug use among urban adolescents placed at risk.

PURPOSE: To examine the relationship between truancy and the onset of drug use. METHODS: Discrete time survival analysis was used to assess the effect of truancy on initiation of drug use after adjusting for several potential confounders from age 11 to 15 years, using data from the Denver Youth Survey, a longitudinal sample of youth who grew up in socially disorganized neighborhoods of Denver, CO. RESULTS: In this population, truancy was a significant predictor of initiation of alcohol, tobacco, and marijuana use. The robust effect of truancy persisted after controlling for potential confounders, including school performance, school isolation, association with delinquent peers, personal delinquent values, parental monitoring, and family attachment. CONCLUSIONS: Although this study cannot point to a causal relationship, we argue that the effect may be at least in part due to the unsupervised, unmonitored time with peers that truancy affords a young person. Truancy prevention is a field of research that needs much more attention. Keeping youth in school every day is likely to have many beneficial effects, and effective truancy prevention efforts may also help to prevent or delay the onset of drug use among adolescents.