Proteomics dataset containing proteins that obscure identification of TOPLESS interactors in Arabidopsis.

Here we report proteins identified after conducting Tandem Affinity Purification (TAP) of the TOPLESS (TPL) corepressor from Arabidopsis. We generated transgenic plants harboring TPL fused to the GS-TAG, "Boosting tandem affinity purification of plant protein complexes" (Van Leene et al., 2008) [1]. Four independent biological replicates of a selected TPL-GS-TAG line were grown simultaneously, crosslinked with formaldehyde, and proteins were isolated from whole plant tissue via TAP. Purified proteins were treated with trypsin, and the peptides were analyzed via mass spectrometry. Datasets are hosted in the MassIVE public repository (reference number: MSV000082477, https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=f16255fb7080426a9fe1926b4d3d5862). The data in this article has not been published elsewhere and is original to this work.

Adaptation of the Agrobacterium tumefaciens VirG response regulator to activate transcription in plants.

The Agrobacterium tumefaciens VirG response regulator of the VirA/VirG two-component system was adapted to function in tobacco protoplasts. The subcellular localization of VirG and VirA proteins transiently expressed in onion cells was determined using GFP fusions. Preliminary studies using Gal4DBD-VP16 fusions with VirG and Escherichia coli UhpA, and NarL response regulators indicated compatibility of these bacterial proteins with the eukaryotic transcriptional apparatus. A strong transcriptional activator based on tandem activation domains from the Drosophila fushi tarazu and Herpes simplex VP16 was created. Selected configurations of the two-site Gal4-vir box GUS reporters were activated by chimeric effectors dependent on either the yeast Gal4 DNA-binding domain or that of VirG. Transcriptional induction of the GUS reporter was highest for the VirE19-element promoter with both constitutive and wild-type VirG-tandem activation domain effectors. Multiple VirE19 elements increased the reporter activity proportionately, indicating that the VirG DNA binding domain was functional in plants. The VirG constitutive-Q-VP16 effector was more active than the VirG wild-type. In both the constitutive and wild-type forms of VirG, Q-VP16 activated transcription of the GUS reporter best when located at the C-terminus, i.e. juxtaposed to the VirG DNA binding domain. These results demonstrate the possibility of using DNA binding domains from bacterial response regulators and their cognate binding elements in the engineering of plant gene expression.

A strategy for building an amplified transcriptional switch to detect bacterial contamination of plants.

We have designed and tested a transcriptional autofeedback loop that could be used to engineer plants to sense the presence of bacteria. The signal amplification circuit was built based on the biological switch responsive to the presence of bacterial flagellin. Several flagellin- and E. coli-inducible Arabidopsis promoters were cloned and tested in transient expression assays in Arabidopsis and lettuce protoplasts using a flagellin-based peptide. These were investigated either as direct drivers of a reporter gene, or as a component of a transcriptional autofeedback loop. Arabidopsis promoters from the xyloglucan endotransglucosylase/hydrolase 18 (ATXTH18) and cytochrome P450 family CYP82C3 monooxygenase worked well as biological switches. These promoters were incorporated into our feedback loop system for signal amplification. The inclusion of a transcriptional repressor reduced basal expression, thereby increasing fold-amplification of signal detection and fine-tuning the positive autofeedback loop regulation.

Parabolic flight induces changes in gene expression patterns in Arabidopsis thaliana.

Our primary objective was to evaluate gene expression changes in Arabidopsis thaliana in response to parabolic flight as part of a comprehensive approach to the molecular biology of spaceflight-related adaptations. In addition, we wished to establish parabolic flight as a tractable operations platform for molecular biology studies. In a succession of experiments on NASA's KC-135 and C-9 parabolic aircraft, Arabidopsis plants were presented with replicated exposure to parabolic flight. Transcriptome profiling revealed that parabolic flight caused changes in gene expression patterns that stood the statistical tests of replication on three different flight days. The earliest response, after 20 parabolas, was characterized by a prominence of genes associated with signal transduction. After 40 parabolas, this prominence was largely replaced by genes associated with biotic and abiotic stimuli and stress. Among these responses, three metabolic processes stand out in particular: the induction of auxin metabolism and signaling, the differential expression of genes associated with calcium-mediated signaling, and the repression of genes associated with disease resistance and cell wall biochemistry. Many, but not all, of these responses are known to be involved in gravity sensing in plants. Changes in auxin-related gene expression were also recorded by reporter genes tuned to auxin signal pathways. These data demonstrate that the parabolic flight environment is appropriate for molecular biology research involving the transition to microgravity, in that with replication, proper controls, and analyses, gene expression changes can be observed in the time frames of typical parabolic flight experiments.

Yeast two-hybrid map of Arabidopsis TFIID.

General transcription factor IID (TFIID) is a multisubunit protein complex involved in promoter recognition and is fundamental to the nucleation of the RNA polymerase II transcriptional preinitiation complex. TFIID is comprised of the TATA binding protein (TBP) and 12-15 TBP-associated factors (TAFs). While general transcription factors have been extensively studied in metazoans and yeast, little is known about the details of their structure and function in the plant kingdom. This work represents the first attempt to compare the structure of a plant TFIID complex with that determined for other organisms. While no TAF3 homolog has been observed in plants, at least one homolog has been identified for each of the remaining 14 TFIID subunits, including both TAF14 and TAF15 which have previously been shown to be unique to either yeast or humans. The presence of both TAFs 14 and 15 in plants suggests ancient roles for these proteins that were lost in metazoans and fungi, respectively. Yeast two-hybrid interaction assays resulted in a total of 65 binary interactions between putative subunits of Arabidopsis TFIID, including 26 contacts unique to plants. The interaction matrix of Arabidopsis TAFs is largely consistent with the three-lobed topological map for yeast TFIID, which suggests that the structure and composition of TFIID have been highly conserved among eukaryotes.

Isolation and characterization of class A4 heat shock transcription factor from alfalfa.

Plant heat shock transcription factors (HSFs) regulate transcription of heat shock (HS) genes. In Arabidopsis thaliana, 21 HSFs have been classified into groups A-C. Members of class A act as typical transcriptional activators, whereas B HSFs function as coactivators or repressors depending on promoter context. The function of class C HSFs is still unclear. Here, we present the isolation and characterization of the first HSF from alfalfa (Medicago sativa L.) and designate it MsHSFA4 based on amino acid sequence analysis. The MsHSFA4 gene was determined to be single copy and was detected at two separate genetic loci in the tetraploid Medicago sativa. Overexpression of MsHSFA4 in tobacco mesophyll protoplasts resulted in weak transcriptional activity, similar to that exhibited by Arabidopsis AtHSFA4a. The MsHSFA4 proximal promoter contains three putative HSE elements, and the gene itself is activated both by heat and cold stress.

Identification and characterization of GIP1, an Arabidopsis thaliana protein that enhances the DNA binding affinity and reduces the oligomeric state of G-box binding factors.

Environmental control of the alcohol dehydrogenase (Adh) and other stress response genes in plants is in part brought about by transcriptional regulation involving the G-box cis-acting DNA element and bZIP G-box Binding Factors (GBFs). The mechanisms of GBF regulation and requirements for additional factors in this control process are not well understood. In an effort to identify potential GBF binding and control partners, maize GBF1 was used as bait in a yeast two-hybrid screen of an A. thaliana cDNA library. GBF Interacting Protein 1 (GIP1) arose from the screen as a 496 amino acid protein with a predicted molecular weight of 53,748 kDa that strongly interacts with GBFs. Northern analysis of A. thaliana tissue suggests a 1.8-1.9 kb GIP1 transcript, predominantly in roots. Immunolocalization studies indicate that GIP1 protein is mainly localized to the nucleus. In vitro electrophoretic mobility shift assays using an Adh G-box DNA probe and recombinant A. thaliana GBF3 or maize GBF1, showed that the presence of GIP1 resulted in a tenfold increase in GBF DNA binding activity without altering the migration, suggesting a transient association between GIP1 and GBF. Addition of GIP1 to intentionally aggregated GBF converted GBF to lower molecular weight macromolecular complexes and GIP1 also refolded denatured rhodanese in the absence of ATP. These data suggest GIP1 functions to enhance GBF DNA binding activity by acting as a potent nuclear chaperone or crowbar, and potentially regulates the multimeric state of GBFs, thereby contributing to bZIP-mediated gene regulation.

Plant class B HSFs inhibit transcription and exhibit affinity for TFIIB and TBP.

Plant heat shock transcription factors (HSFs) are capable of transcriptional activation (class A HSFs) or both, activation and repression (class B HSFs). However, the details of mechanism still remain unclear. It is likely, that the regulation occurs through interactions of HSFs with general transcription factors (GTFs), as has been described for numerous other transcription factors. Here, we show that class A HSFs may activate transcription through direct contacts with TATA-binding protein (TBP). Class A HSFs can also interact weakly with TFIIB. Conversely, class B HSFs inhibit promoter activity through an active mechanism of repression that involves the C-terminal regulatory region (CTR) of class B HSFs. Deletion analysis revealed two sites in the CTR of soybean GmHSFB1 potentially involved in protein-protein interactions with GTFs: one is the repressor domain (RD) located in the N-terminal half of the CTR, and the other is a TFIIB binding domain (BD) that shows affinity for TFIIB and is located C-terminally from the RD. A Gal4 DNA binding domain-RD fusion repressed activity of LexA-activators, while Gal4-BD proteins synergistically activated strong and weak transcriptional activators. In vitro binding studies were consistent with this pattern of activity since the BD region alone interacted strongly with TFIIB, and the presence of RD had an inhibitory effect on TFIIB binding and transcriptional activation.

Plant adaptation to low atmospheric pressures: potential molecular responses.

There is an increasing realization that it may be impossible to attain Earth normal atmospheric pressures in orbital, lunar, or Martian greenhouses, simply because the construction materials do not exist to meet the extraordinary constraints imposed by balancing high engineering requirements against high lift costs. This equation essentially dictates that NASA have in place the capability to grow plants at reduced atmospheric pressure. Yet current understanding of plant growth at low pressures is limited to just a few experiments and relatively rudimentary assessments of plant vigor and growth. The tools now exist, however, to make rapid progress toward understanding the fundamental nature of plant responses and adaptations to low pressures, and to develop strategies for mitigating detrimental effects by engineering the growth conditions or by engineering the plants themselves. The genomes of rice and the model plant Arabidopsis thaliana have recently been sequenced in their entirety, and public sector and commercial DNA chips are becoming available such that thousands of genes can be assayed at once. A fundamental understanding of plant responses and adaptation to low pressures can now be approached and translated into procedures and engineering considerations to enhance plant growth at low atmospheric pressures. In anticipation of such studies, we present here the background arguments supporting these contentions, as well as informed speculation about the kinds of molecular physiological responses that might be expected of plants in low-pressure environments.