Auxin signal transduction.

Abstract The plant hormone auxin (indole-3-acetic acid, IAA) controls growth and developmental responses throughout the life of a plant. A combination of molecular, genetic and biochemical approaches has identified several key components involved in auxin signal transduction. Rapid auxin responses in the nucleus include transcriptional activation of auxin-regulated genes and degradation of transcriptional repressor proteins. The nuclear auxin receptor is an integral component of the protein degradation machinery. Although auxin signalling in the nucleus appears to be short and simple, recent studies indicate that there is a high degree of diversity and complexity, largely due to the existence of multigene families for each of the major molecular components. Current studies are attempting to identify interacting partners among these families, and to define the molecular mechanisms involved in the interactions. Future goals are to determine the levels of regulation of the key components of the transcriptional complex, to identify higher-order complexes and to integrate this pathway with other auxin signal transduction pathways, such as the pathway that is activated by auxin binding to a different receptor at the outer surface of the plasma membrane. In this case, auxin binding triggers a signal cascade that affects a number of rapid cytoplasmic responses. Details of this pathway are currently under investigation

Study of auxin responsive gene promoters in transient expression assays

Abstract only availableTransfection assays are an important tool for biochemists; the comparatively quick technique allows experimenters to see relative levels of expression of a particular gene in the presence or absence of a plant hormone and/or effector. Transfection assays can be used to study the promoter region of a gene to determine the importance of specific cis-regulatory elements in the control of gene expression. This is accomplished by fusing the promoter region with a reporter gene. A reporter gene allows the expression level of the promoter to be monitored by fluorescence or by other observable properties. Transfection can also be used to study growth substances and various cellular signaling pathways. This technique involves isolating protoplasts from cells such as carrot suspension cells. In order to use these suspension cells, the cell walls must be digested; thus the cells become protoplasts. The experimental DNA (effector and reporter genes) is then introduced into protoplasts by chemical methods. The protoplasts are incubated in the dark and the relative reporter gene activity is then read on a fluorometer. The goal of this project is to determine if two Aux/IAA genes are auxin inducible and what influence specific effectors have on their expression using carrot protoplasts to do transient transfection assays. We study auxin responsive gene promoters because many of them contain auxin responsive cis-regulatory elements designated as Auxin Response Elements (AuxREs), which contain the sequence TGTCTC. These promoters are regulated by at least two groups of transcription factors known as Auxin Response Factors (ARFs) and Aux/IAA proteins. One group of auxin inducible genes is Aux/IAA genes. We studied the Aux/IAA17 and Aux/IAA19 promoter regions by amplifying them through PCR and cloning them upstream of the ß-glucuronidase (GUS) reporter gene open reading frame (ORF). This reporter gene was used because the GUS activity can be detected through fluorescence. The GUS gene was terminated by a nopaline synthase (NOS) terminator cloned downstream of the GUS gene. The effectors used in these studies consisted of ARF proteins translated from ARF effector genes during the transfection process. Results showed that the Aux/IAA19 gene (which has 3 cis-regulatory AuxREs) was inducible by auxin. The ARF effectors influenced the expression of both Aux/IAA genes by positively or negatively regulating their expression. However, auxin was found to have no affect on Aux/IAA17 gene expression. This correlates with the fact that Aux/IAA17 has no AuxREs in its promoter region.Plant Genomics Internship @ M

Inference of the Arabidopsis Lateral Root Gene Regulatory Network Suggests a Bifurcation Mechanism That Defines Primordia Flanking and Central Zones

A large number of genes involved in lateral root (LR) organogenesis have been identified over the last decade using forward and reverse genetic approaches in Arabidopsis thaliana. Nevertheless, how these genes interact to form a LR regulatory network largely remains to be elucidated. In this study, we developed a time-delay correlation algorithm (TDCor) to infer the gene regulatory network (GRN) controlling LR primordium initiation and patterning in Arabidopsis from a time-series transcriptomic data set. The predicted network topology links the very early-activated genes involved in LR initiation to later expressed cell identity markers through a multistep genetic cascade exhibiting both positive and negative feedback loops. The predictions were tested for the key transcriptional regulator AUXIN RESPONSE FACTOR7 node, and over 70% of its targets were validated experimentally. Intriguingly, the predicted GRN revealed a mutual inhibition between the ARF7 and ARF5 modules that would control an early bifurcation between two cell fates. Analyses of the expression pattern of ARF7 and ARF5 targets suggest that this patterning mechanism controls flanking and central zone specification in Arabidopsis LR primordia

AUXIN RESPONSE FACTOR1 and AUXIN RESPONSE FACTOR2regulate senescence and floral organ abscission in Arabidopsisthaliana

In plants, both endogenous mechanisms and environmental signals regulate developmental transitions such as seed germination, induction of flowering, leaf senescence and shedding of senescent organs. Auxin response factors (ARFs) are transcription factors that mediate responses to the plant hormone auxin. We have examine

Treaties in Collision: The Biosafety Protocol and the World Trade Organization Agreements

In the event of a conflict between the requirements of the Biosafety Protocol, a multilateral agreement governing the trade in genetically modified organisms, and the requirements of the General Agreement on Tariffs and Trade and associated agreements (collectively WTO Agreements), which treaty\u27s requirements prevail? This question lies as the legal heart of the perceived conflict between trade globalization and environmental protection. This issue is particularly timely given the present trade dispute between the United States and European Union over the European Union’s restrictions on the importation of genetically modified agricultural commodities. In this piece, I analyze the relationship between these agreements. I conclude that while the “savings clause” language ultimately included in the Biosafety Protocol preserves countries’ rights and obligations under the WTO Agreements, the Protocol and the WTO Agreements are less on a collision course than some may fear

Auxin response factors ARF6 and ARF8 promote jasmonic acid production and flower maturation

Pollination in flowering plants requires that anthers release pollen when the gynoecium is competent to support fertilization. We show that i

Cloning auxin-responsive genes for future study in Arabidopsis [abstract]

Abstract only availableFaculty Mentor: Dr. Gretchen Hagen, BiochemistryAuxin, a plant hormone, serves as a signal for cells to grow, divide, and differentiate. Approximately 25 years ago, auxin was shown to rapidly affect the expression of specific genes. To date, only three families of auxin responsive genes have been extensively characterized: Aux/IAA, GH3, and SAUR. However, newly published data from another lab has identified other auxin-regulated genes in Arabidopsis thaliana. The purpose of this project was to clone the open reading frames (ORFs) of six of these genes into vectors for over-expression in Arabidopsis. To clone the ORFs of the six genes, primers containing their start and stop codons and restriction sites were designed and used in a reverse transcription polymerase chain reaction (RT-PCR) to amplify the ORFs. RT-PCR was carried out by isolating RNA from Arabidopsis tissue culture and leaf RNA and synthesizing complimentary DNA (cDNA) using oligo dT and reverse transcriptase. The primers were used in a PCR reaction to amplify the ORFs in the cDNA. RT-PCR products were purified and digested at the designed restriction sites. The ORFs were fused to the strong, constituitive 35S CaMV promoter in pUC19, the constructs were transformed into DH5α E. coli cells, and antibiotic resistant colonies were selected. Colonies were screened by PCR using insert-specific primers. Those identified to have the insert by agarose gel electrophoresis were grown in liquid media, so their DNA could be isolated and analyzed by sequencing. The inserts were then cloned into the pPZP211 (an Agrobacterium binary vector), and transformed into DH5α. Cells containing the construct were used in tri-parental mating with Agrobacterium tumefaciens and DH5α RK2013 (containing a helper plasmid). Arabidopsis was vacuum infiltrated using transformed Agrobacterium. Seed of infiltrated plants will be screened to obtain transformed plant lines. Ten to twenty lines will be examined for over-expression of the ORF and any unusual phenotypes. Currently, three of the six constructs have been infiltrated into Arabidopsis. A fourth construct has gone through tri-parental mating, and is in Agrobacterium. The other two constructs are in different stages of the cloning process