Online Science Instruction Can Promote Adolescents' Autonomy Need Satisfaction: a Latent Growth Curve Analysis.

This research examined the differential motivational effects of a pre-college science enrichment program delivered in both online and in-person learning formats. Using self-determination theory as a guiding framework, we hypothesized that (a) students would exhibit growth in their perceived satisfaction of needs for autonomy, competence, and relatedness, (b) online learning would be associated with greater growth in autonomy, and (c) in-person learning would be associated with greater growth in both competence and relatedness. Using a sample of 598 adolescent participants, results of latent growth curve modeling indicated that satisfaction of the three needs grew unconditionally over the course of the program. However, format type was unrelated to growth in need satisfaction. Rather, this effect was found to be conditional upon the type of science project undertaken by students: astrophysics students exhibited significantly greater autonomy growth when receiving online instruction than did biochemistry students. Our findings suggest that online science learning can be just as effective in motivating students as in-person learning provided that the learning tasks are conducive to remote instruction.

CHD Chromatin Remodeling Protein Diversification Yields Novel Clades and Domains Absent in Classic Model Organisms.

Chromatin remodelers play a fundamental role in the assembly of chromatin, regulation of transcription, and DNA repair. Biochemical and functional characterizations of the CHD family of chromatin remodelers from a variety of model organisms have shown that these remodelers participate in a wide range of activities. However, because the evolutionary history of CHD homologs is unclear, it is difficult to predict which of these activities are broadly conserved and which have evolved more recently in individual eukaryotic lineages. Here, we performed a comprehensive phylogenetic analysis of 8,042 CHD homologs from 1,894 species to create a model for the evolution of this family across eukaryotes with a particular focus on the timing of duplications that gave rise to the diverse copies observed in plants, animals, and fungi. Our analysis confirms that the three major subfamilies of CHD remodelers originated in the eukaryotic last common ancestor, and subsequent losses occurred independently in different lineages. Improved taxon sampling identified several subfamilies of CHD remodelers in plants that were absent or highly divergent in the model plant Arabidopsis thaliana. Whereas the timing of CHD subfamily expansions in vertebrates corresponds to whole genome duplication events, the mechanisms underlying CHD diversification in land plants appear more complicated. Analysis of protein domains reveals that CHD remodeler diversification has been accompanied by distinct transitions in domain architecture, contributing to the functional differences observed between these remodelers. This study demonstrates the importance of proper taxon sampling when studying ancient evolutionary events to prevent misinterpretation of subsequent lineage-specific changes and provides an evolutionary framework for functional and comparative analysis of this critical chromatin remodeler family across eukaryotes.

Overexpression of arogenate dehydratase reveals an upstream point of metabolic control in phenylalanine biosynthesis.

Out of the three aromatic amino acids, the highest flux in plants is directed towards phenylalanine, which is utilized to synthesize proteins and thousands of phenolic metabolites contributing to plant fitness. Phenylalanine is produced predominantly in plastids via the shikimate pathway and subsequent arogenate pathway, both of which are subject to complex transcriptional and post-transcriptional regulation. Previously, it was shown that allosteric feedback inhibition of arogenate dehydratase (ADT), which catalyzes the final step of the arogenate pathway, restricts flux through phenylalanine biosynthesis. Here, we show that in petunia (Petunia hybrida) flowers, which typically produce high phenylalanine levels, ADT regulation is relaxed, but not eliminated. Moderate expression of a feedback-insensitive ADT increased flux towards phenylalanine, while high overexpression paradoxically reduced phenylalanine formation. This reduction could be partially, but not fully, recovered by bypassing other known metabolic flux control points in the aromatic amino acid network. Using comparative transcriptomics, reverse genetics, and metabolic flux analysis, we discovered that transcriptional regulation of the d-ribulose-5-phosphate 3-epimerase gene in the pentose phosphate pathway controls flux into the shikimate pathway. Taken together, our findings reveal that regulation within and upstream of the shikimate pathway shares control over phenylalanine biosynthesis in the plant cell.

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio.

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development of a customizable platform to rapidly generate gene modifications in a wide variety of organisms, including zebrafish. CRISPR-based genome editing uses a single guide RNA (sgRNA) to target a CRISPR-associated (Cas) endonuclease to a genomic DNA (gDNA) target of interest, where the Cas endonuclease generates a double-strand break (DSB). Repair of DSBs by error-prone mechanisms lead to insertions and/or deletions (indels). This can cause frameshift mutations that often introduce a premature stop codon within the coding sequence, thus creating a protein-null allele. CRISPR-based genome engineering requires only a few molecular components and is easily introduced into zebrafish embryos by microinjection. This protocol describes the methods used to generate CRISPR reagents for zebrafish microinjection and to identify fish exhibiting germline transmission of CRISPR-modified genes. These methods include in vitro transcription of sgRNAs, microinjection of CRISPR reagents, identification of indels induced at the target site using a PCR-based method called a heteroduplex mobility assay (HMA), and characterization of the indels using both a low throughput and a powerful next-generation sequencing (NGS)-based approach that can analyze multiple PCR products collected from heterozygous fish. This protocol is streamlined to minimize both the number of fish required and the types of equipment needed to perform the analyses. Furthermore, this protocol is designed to be amenable for use by laboratory personal of all levels of experience including undergraduates, enabling this powerful tool to be economically employed by any research group interested in performing CRISPR-based genomic modification in zebrafish.

Regulation of membrane fatty acid composition by temperature in mutants of Arabidopsis with alterations in membrane lipid composition.

BACKGROUND: A wide range of cellular responses occur when plants are exposed to elevated temperature, including adjustments in the unsaturation level of membrane fatty acids. Although membrane bound desaturase enzymes mediate these adjustments, it is unknown how they are regulated to achieve these specific membrane compositions. Furthermore, the precise roles that different membrane fatty acid compositions play in photosynthesis are only beginning to be understood. To explore the regulation of the membrane composition and photosynthetic function in response to temperature, we examined the effect of temperature in a collection of mutants with altered membrane lipid fatty acid composition. RESULTS: In agreement with previous studies in other species, the level of unsaturation of membrane fatty acids in Arabidopsis was inversely correlated with growth temperature. The time required for the membrane fatty acids to attain the composition observed at elevated temperature was consistent with the timing required for the synthesis of new fatty acids. Comparisons of temperature-induced fatty acid alterations in membranes were made among several Arabidopsis lines including wild-type Columbia, and the compositional mutants, fad5, fad6, act1 and double mutants, fad7 fad8 and act1 fad6. The results revealed key changes that occur in response to elevated temperature regardless of the specific mutations in the glycerolipid pathway, including marked decreases in trienoic fatty acids and consistent increases in unsaturated 16:0 and in dienoic 18:2 levels. Fluorescence measurements of various mutants indicated that photosynthetic stability as well as whole plant growth at elevated temperature is influenced by certain membrane fatty acid compositions. CONCLUSIONS: The results of this study support the premise that defined proportions of saturated and unsaturated fatty acids in membrane lipids are required for photosynthetic thermostability and acclimation to elevated temperature. The results also suggest that changes in the membrane fatty acid composition brought about in response to temperature are regulated in such a way so as to achieve highly similar unsaturation levels despite mutations that alter the membrane composition prior to a high-temperature exposure. The results from examination of the mutant lines also suggest that interorganellar transfer of fatty acids are involved in mediating temperature-induced membrane alterations, and reveal steps in the fatty acid unsaturation pathway that appear to have key roles in the acclimatization of membranes to high temperature.

Light induces phenylpropanoid metabolism in Arabidopsis roots.

Experiments have shown that many phenylpropanoid genes are highly expressed in light-grown Arabidopsis roots. Studies employing reporter gene constructs have indicated that the expression of these genes is localized not only to the lignifying root vasculature, but also to non-lignifying tissues, such as the root cortex, suggesting that the proteins encoded by these genes may be involved in aspects of phenylpropanoid metabolism other than lignification. Consistent with this hypothesis, roots of etiolated and soil-grown plants contain almost no soluble phenylpropanoids, but exposure to light leads to the accumulation of flavonoids, as well as high levels of coniferin and syringin (coniferyl and sinapyl-4-O-glycosides), compounds not previously reported to be accumulated in Arabidopsis. To elucidate the mechanism by which light induces root secondary metabolism, extracts of mutants defective in light perception and light responses were analyzed for phenylpropanoid content. The results of these assays showed that phytochrome (PHY)B and cryptochrome (CRY)2 are the primary photoreceptors involved in light-dependent phenylpropanoid accumulation, and that the hypocotyl elongated (HY5) transcription factor is also required for this response. The presence of phenylpropanoids in etiolated roots of cop (constitutively photomorphogenic)1, cop9, and det (de-etiolated)1 mutants indicate that the corresponding wild-type genes are required to repress root phenylpropanoid biosynthesis in the absence of light. Biochemical analysis of root cell walls and analysis of phenylpropanoid gene expression suggest that coniferin and syringin accumulation may be the result of both increased biosynthesis and decreased conversion of these compounds into other phenylpropanoid end products. Finally, our data suggest that the accumulation of coniferin, syringin, and flavonoids in Arabidopsis roots is a high-irradiance response (HIR), and suggest that comparative analysis of light- and dark-grown Arabidopsis roots may provide new insights into both phenylpropanoid biosynthesis and light signaling in plants.