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Abstract PremiseA multi‐omic approach was used to explore proteins and networks hypothetically important for establishing filament dimorphisms in heterostylousTurnera subulata(Sm.) as an exploratory method to identify genes for future empirical research. MethodsMass spectrometry (MS) was used to identify differentially expressed proteins and differentially phosphorylated peptides in the developing filaments between the L‐ and S‐morphs. RNAseq was used to generate a co‐expression network of the developing filaments, MS data were mapped to the co‐expression network to identify hypothetical relationships between theS‐gene responsible for filament dimorphisms and differentially expressed proteins. ResultsMapping all MS identified proteins to a co‐expression network of the S‐morph's developing filaments identified several clusters containing SPH1 and other differentially expressed or phosphorylated proteins. Co‐expression analysis clustered CDKG2, a protein that induces endoreduplication, and SPH1—suggesting a shared biological function. MS analysis suggests that the protein is present and phosphorylated only in the S‐morph, and thus active only in the S‐morph. A series of CDKG2 regulators, including ATM1, and cell cycle regulators also correlated with the presence of reciprocal herkogamy, supporting our interest in the protein. ConclusionsThis work has built a foundation for future empirical work, specifically supporting the role of CDKG2 and ATM1 in promoting filament elongation in response to SPH1 perception. 

Abstract Maintenance of water homeostasis is a fundamental cellular process required by all living organisms. Here, we use the single-celled green algaChlamydomonas reinhardtiito establish a foundational understanding of osmotic-stress signaling pathways through transcriptomics, phosphoproteomics, and functional genomics approaches. Comparison of pathways identified through these analyses with yeast and Arabidopsis allows us to infer their evolutionary conservation and divergence across these lineages. 76 genes, acting across diverse cellular compartments, were found to be important for osmotic-stress tolerance in Chlamydomonas through their functions in cytoskeletal organization, potassium transport, vesicle trafficking, mitogen-activated protein kinase and chloroplast signaling. We show that homologs for five of these genes have conserved functions in stress tolerance in Arabidopsis and reveal a novel PROFILIN-dependent stage of acclimation affecting the actin cytoskeleton that ensures tissue integrity upon osmotic stress. This study highlights the conservation of the stress response in algae and land plants, and establishes Chlamydomonas as a unicellular plant model system to dissect the osmotic stress signaling pathway. 

Abstract Over the past two decades, mass spectrometric (MS)-based proteomics technologies have facilitated the study of signaling pathways throughout biology. Nowhere is this needed more than in plants, where an evolutionary history of genome duplications has resulted in large gene families involved in posttranslational modifications and regulatory pathways. For example, at least 5% of the Arabidopsis thaliana genome (ca. 1,200 genes) encodes protein kinases and protein phosphatases that regulate nearly all aspects of plant growth and development. MS-based technologies that quantify covalent changes in the side-chain of amino acids are critically important, but they only address one piece of the puzzle. A more crucially important mechanistic question is how noncovalent interactions—which are more difficult to study—dynamically regulate the proteome’s 3D structure. The advent of improvements in protein 3D technologies such as cryo-electron microscopy, nuclear magnetic resonance, and X-ray crystallography has allowed considerable progress to be made at this level, but these methods are typically limited to analyzing proteins, which can be expressed and purified in milligram quantities. Newly emerging MS-based technologies have recently been developed for studying the 3D structure of proteins. Importantly, these methods do not require protein samples to be purified and require smaller amounts of sample, opening the wider proteome for structural analysis in complex mixtures, crude lysates, and even in intact cells. These MS-based methods include covalent labeling, crosslinking, thermal proteome profiling, and limited proteolysis, all of which can be leveraged by established MS workflows, as well as newly emerging methods capable of analyzing intact macromolecules and the complexes they form. In this review, we discuss these recent innovations in MS-based “structural” proteomics to provide readers with an understanding of the opportunities they offer and the remaining challenges for understanding the molecular underpinnings of plant structure and function.