An optimized pipeline for live imaging whole Arabidopsis leaves at cellular resolution.

BACKGROUND: Live imaging is the gold standard for determining how cells give rise to organs. However, tracking many cells across whole organs over large developmental time windows is extremely challenging. In this work, we provide a comparably simple method for confocal live imaging entire Arabidopsis thaliana first leaves across early development. Our imaging method works for both wild-type leaves and the complex curved leaves of the jaw-1D mutant. RESULTS: We find that dissecting the cotyledons, affixing a coverslip above the samples and mounting samples with perfluorodecalin yields optimal imaging series for robust cellular and organ level analysis. We provide details of our complementary image processing steps in MorphoGraphX software for segmenting, tracking lineages, and measuring a suite of cellular properties. We also provide MorphoGraphX image processing scripts we developed to automate analysis of segmented images and data presentation. CONCLUSIONS: Our imaging techniques and processing steps combine into a robust imaging pipeline. With this pipeline we are able to examine important nuances in the cellular growth and differentiation of jaw-D versus WT leaves that have not been demonstrated before. Our pipeline is approachable and easy to use for leaf development live imaging.

Modification of G-protein biochemistry and its effect on plant/environment interaction.

Heterotrimeric GTP-binding proteins comprised of Gα, Gß and Gγ subunits are key regulators of a multitude of signaling pathways in eukaryotes. In plants, G-proteins are currently a focus of intense research due to their involvement in modulation of many agronomically important traits such as seed yield, organ size, abscisic acid (ABA)-dependent signaling and stress responses, plant defense responses, symbiosis and nitrogen use efficiency. The mechanistic details of G-protein biochemistry in modulating these processes in plants remain largely unknown. Although the core G-protein components and their activation/deactivation chemistries are broadly conserved throughout eukaryotic evolution, their regulation seems to have been rewired in plants to meet specific needs. Plant G-proteins may be spontaneously active and/or are regulated by phosphorylation-dependent changes, by the activity of lipid second messengers such as phospholipases, or may even have nucleotide-exchange independent regulation. Regardless of these deviations from the established norm, the biochemical properties of plant G-proteins are key to affecting plant phenotypes and responses. Detailed characterization of such activities, in vitro and in planta, will pave the way for precise manipulation of these proteins for future agricultural needs.

A Life Cycle for Modeling Biology at Different Scales.

Modeling has become a popular tool for inquiry and discovery across biological disciplines. Models allow biologists to probe complex questions and to guide experimentation. Modeling literacy among biologists, however, has not always kept pace with the rise in popularity of these techniques and the relevant advances in modeling theory. The result is a lack of understanding that inhibits communication and ultimately, progress in data gathering and analysis. In an effort to help bridge this gap, we present a blueprint that will empower biologists to interrogate and apply models in their field. We demonstrate the applicability of this blueprint in two case studies from distinct subdisciplines of biology; developmental-biomechanics and evolutionary biology. The models used in these fields vary from summarizing dynamical mechanisms to making statistical inferences, demonstrating the breadth of the utility of models to explore biological phenomena.

Plant Morphogenesis: Mechanical Feedback Position Is Crucial in Organ Flattening.

A new study presents a three-dimensional mechanical model with multiple cell layers to interrogate the flattening of organs during development. This model shows the importance of initial asymmetry and its reinforcement by mechanical feedback within the inner cell walls, not the outer epidermal wall, in guiding organ flattening of organ primordia.

Maize Introgression Library Provides Evidence for the Involvement of liguleless1 in Resistance to Northern Leaf Blight.

Plant disease resistance is largely governed by complex genetic architecture. In maize, few disease resistance loci have been characterized. Near-isogenic lines are a powerful genetic tool to dissect quantitative trait loci. We analyzed an introgression library of maize (Zea mays) near-isogenic lines, termed a nested near-isogenic line library for resistance to northern leaf blight caused by the fungal pathogen Setosphaeria turcica The population was comprised of 412 BC5F4 near-isogenic lines that originated from 18 diverse donor parents and a common recurrent parent, B73. Single nucleotide polymorphisms identified through genotyping by sequencing were used to define introgressions and for association analysis. Near-isogenic lines that conferred resistance and susceptibility to northern leaf blight were comprised of introgressions that overlapped known northern leaf blight quantitative trait loci. Genome-wide association analysis and stepwise regression further resolved five quantitative trait loci regions, and implicated several candidate genes, including Liguleless1, a key determinant of leaf architecture in cereals. Two independently-derived mutant alleles of liguleless1 inoculated with S. turcica showed enhanced susceptibility to northern leaf blight. In the maize nested association mapping population, leaf angle was positively correlated with resistance to northern leaf blight in five recombinant inbred line populations, and negatively correlated with northern leaf blight in four recombinant inbred line populations. This study demonstrates the power of an introgression library combined with high density marker coverage to resolve quantitative trait loci. Furthermore, the role of liguleless1 in leaf architecture and in resistance to northern leaf blight has important applications in crop improvement.

The plant pathogen enzyme AldC is a long-chain aliphatic aldehyde dehydrogenase.

Aldehyde dehydrogenases are versatile enzymes that serve a range of biochemical functions. Although traditionally considered metabolic housekeeping enzymes because of their ability to detoxify reactive aldehydes, like those generated from lipid peroxidation damage, the contributions of these enzymes to other biological processes are widespread. For example, the plant pathogen Pseudomonas syringae strain PtoDC3000 uses an indole-3-acetaldehyde dehydrogenase to synthesize the phytohormone indole-3-acetic acid to elude host responses. Here we investigate the biochemical function of AldC from PtoDC3000. Analysis of the substrate profile of AldC suggests that this enzyme functions as a long-chain aliphatic aldehyde dehydrogenase. The 2.5 Å resolution X-ray crystal of the AldC C291A mutant in a dead-end complex with octanal and NAD+ reveals an apolar binding site primed for aliphatic aldehyde substrate recognition. Functional characterization of site-directed mutants targeting the substrate- and NAD(H)-binding sites identifies key residues in the active site for ligand interactions, including those in the "aromatic box" that define the aldehyde-binding site. Overall, this study provides molecular insight for understanding the evolution of the prokaryotic aldehyde dehydrogenase superfamily and their diversity of function.