An Inquiry-Infused Introductory Biology Laboratory That Integrates Mendel\u27s Pea Phenotypes With Molecular Mechanisms

We developed a multi-week laboratory in which college-level introductory biology students investigate Mendel\u27s stem length phenotype in peas. Students collect, analyze and interpret convergent evidence from molecular and physiological techniques. In weeks 1 and 2, students treat control and experimental plants with Gibberellic Acid (GA) to determine whether uncharacterized short mutant lines are GA responsive. These data allow students to place the mutation in the GA signal transduction pathway. During weeks 2 and 3, plants are genotyped for Mendel\u27s le mutation using a derived cleaved polymorphic sequences (dCAPS) PCR assay. This laboratory allows students to make a direct connection between modern molecular genetics and the easily scored phenotypes Mendel used as the basis of his fundamental discoveries. We administered surveys to assess student gains in accord with four learning goals: understanding the lab, basic science literacy, scientific practices, and working collaboratively. Student confidence increased significantly in the first three, but not in working collaboratively, although students reported greater confidence working in groups than alone

Some Like It Hot, Some Like It Warm: Phenotyping To Explore Thermotolerance Diversity

Plants have evolved overlapping but distinct cellular responses to different aspects of high temperature stress. These responses include basal thermotolerance, short- and long-term acquired thermotolerance, and thermotolerance to moderately high temperatures. This ‘thermotolerance diversity’ means that multiple phenotypic assays are essential for fully describing the functions of genes involved in heat stress responses. A large number of genes with potential roles in heat stress responses have been identified using genetic screens and genome wide expression studies. We examine the range of phenotypic assays that have been used to characterize thermotolerance phenotypes in both Arabidopsis and crop plants. Three major variables differentiate thermotolerance assays: (1) the heat stress regime used, (2) the developmental stage of the plants being studied, and (3) the actual phenotype which is scored. Consideration of these variables will be essential for deepening our understanding of the molecular genetics of plant thermotolerance

\u3cem\u3eHSFA1d\u3c/em\u3e Regulates The Kinetics Of Heat-Induced \u3cem\u3eHSP17.6\u3c/em\u3e Expression In Arabidopsis

Arabidopsis contains four HSFA1 heat shock transcription factors (HSFA1a, HSFA1b, HSFA1d, and HSFA1e) that regulate the primary response to high temperature stress responses. These genes have overlapping functions and, while double and triple HSFA1 mutants have thermotolerance phenotypes, these genes have no reported single mutant thermotolerance phenotypes. We used an automated fluorescence microscopy system to quantitate the expression of a HSP17.6:GFP reporter with high temporal resolution to show that HSFA1d is required for normal heat-induced HSP17.6 expression. HSP17.6 expression is reduced and delayed in hsfa1d-1 mutants. This finding highlights the power of using gene expression kinetics as a quantitative phenotype for discovering the function of genes that exhibit functional redundancy

The RootScope: A Simple High-Throughput Screening System For Quantitating Gene Expression Dynamics In Plant Roots

Background: High temperature stress responses are vital for plant survival. The mechanisms that plants use to sense high temperatures are only partially understood and involve multiple sensing and signaling pathways. Here we describe the development of the RootScope, an automated microscopy system for quantitating heat shock responses in plant roots.Results: The promoter of Hsp17.6 was used to build a Hsp17.6(p):GFP transcriptional reporter that is induced by heat shock in Arabidopsis. An automated fluorescence microscopy system which enables multiple roots to be imaged in rapid succession was used to quantitate Hsp17.6p: GFP response dynamics. Hsp17.6(p):GFP signal increased with temperature increases from 28 degrees C to 37 degrees C. At 40 degrees C the kinetics and localization of the response are markedly different from those at 37 degrees C. This suggests that different mechanisms mediate heat shock responses above and below 37 degrees C. Finally, we demonstrate that Hsp17.6(p):GFP expression exhibits wave like dynamics in growing roots.Conclusions: The RootScope system is a simple and powerful platform for investigating the heat shock response in plants

Genetic Interactions Between BOB1 And Multiple 26S Proteasome Subunits Suggest A Role For Proteostasis In Regulating Arabidopsis Development

Protein folding and degradation are both required for protein quality control, an essential cellular activity that underlies normal growth and development. We investigated how BOB1, an Arabidopsis thaliana small heat shock protein, maintains normal plant development. bob1 mutants exhibit organ polarity defects and have expanded domains of KNOX gene expression. Some of these phenotypes are ecotype specific suggesting that other genes function to modify them. Using a genetic approach we identified an interaction between BOB1 and FIL, a gene required for abaxial organ identity. We also performed an EMS enhancer screen using the bob1-3 allele to identify pathways that are sensitized by a loss of BOB1 function. This screen identified genetic, but not physical, interactions between BOB1 and the proteasome subunit RPT2a. Two other proteasome subunits, RPN1a and RPN8a, also interact genetically with BOB1. Both BOB1 and the BOB1-interacting proteasome subunits had previously been shown to interact genetically with the transcriptional enhancers AS1 and AS2, genes known to regulate both organ polarity and KNOX gene expression. Our results suggest a model in which BOB1 mediated protein folding and proteasome mediated protein degradation form a functional proteostasis module required for ensuring normal plant development

The Embryonic Transcriptome Of The Red-Eared Slider Turtle (Trachemys Scripta)

The bony shell of the turtle is an evolutionary novelty not found in any other group of animals, however, research into its formation has suggested that it has evolved through modification of conserved developmental mechanisms. Although these mechanisms have been extensively characterized in model organisms, the tools for characterizing them in non-model organisms such as turtles have been limited by a lack of genomic resources. We have used a next generation sequencing approach to generate and assemble a transcriptome from stage 14 and 17 Trachemys scripta embryos, stages during which important events in shell development are known to take place. The transcriptome consists of 231,876 sequences with an N-50 of 1,166 bp. GO terms and EC codes were assigned to the 61,643 unique predicted proteins identified in the transcriptome sequences. All major GO categories and metabolic pathways are represented in the transcriptome. Transcriptome sequences were used to amplify several cDNA fragments designed for use as RNA in situ probes. One of these, BMP5, was hybridized to a T. scripta embryo and exhibits both conserved and novel expression patterns. The transcriptome sequences should be of broad use for understanding the evolution and development of the turtle shell and for annotating any future T. scripta genome sequences

Temperature compensation of auxin dependent developmental patterning

The establishment of localized auxin gradients plays a central role in developmental patterning in plants. Auxin levels and responses have been shown to increase with temperature although developmental patterning is not affected. This suggests the existence of a homeostatic mechanism that ensures that patterning occurs normally over a range of temperatures. We recently described the cloning and characterization of BOBBER1 (BOB1), an Arabidopsis gene which encodes a small heat shock protein. BOB1 is required for the establishment of auxin gradients and for normal developmental patterning. BOB1 is also required for organismal thermotolerance and localizes to heat shock granules at elevated temperatures. Since BOB1 functions in both temperature responses and developmental patterning we propose that BOB1 may encode a component of a developmental temperature compensation mechanism