Justin W. Walley

Reconstruction of protein networks from an atlas of maize seed proteotypes

Significance Here we report deep, quantitative, and replicated proteome analysis of a developing multicellular organism. We quantified protein abundance and levels of protein phosphorylation during development of the maize seed. The depth and quantitative nature of the data enabled a network-based approach to identify kinase-substrate relationships as well as the reconstruction of biochemical and signaling networks that underpin seed development and seed storage product production. We found that many of the most abundant proteins are not associated with detectable levels of their mRNAs and vice versa. These data significantly add to our understanding of seed development and facilitate knowledge-based crop improvement. A comprehensive knowledge of proteomic states is essential for understanding biological systems. Using mass spectrometry, we mapped an atlas of developing maize seed proteotypes comprising 14,165 proteins and 18,405 phosphopeptides (from 4,511 proteins), quantified across eight tissues. We found that many of the most abundant proteins are not associated with detectable levels of their mRNAs, and we provide evidence for three potential explanations: transport of proteins between tissues; diurnal, out-of-phase accumulation of mRNAs and cognate proteins; and differential lifetimes of mRNAs compared with proteins. Likewise, many of the most abundant mRNAs were not associated with detectable levels of their proteins. Across the entire dataset, protein abundance was poorly correlated with mRNA levels and was largely independent of phosphorylation status. Comparisons between proteotypes revealed the quantitative contribution of specific proteins and phosphorylation events to the spatially and temporally regulated starch and oil biosynthetic pathways. Reconstruction of signaling networks established associations of proteins and phosphoproteins with distinct biological processes acting during seed development. Additionally, a protein kinase substrate network was reconstructed, enabling the identification of 762 potential substrates of specific protein kinases. Finally, examination of 694 transcription factors revealed remarkable constraints on patterns of expression and phosphorylation within transcription factor families. These results provide a resource for understanding seed development in a crop that is the foundation of modern agriculture.

An Automated Proteogenomic Method Uses Mass Spectrometry to Reveal Novel Genes in Zea mays

New technologies in genomics and proteomics have influenced the emergence of proteogenomics, a field at the confluence of genomics, transcriptomics, and proteomics. First generation proteogenomic toolkits employ peptide mass spectrometry to identify novel protein coding regions. We extend first generation proteogenomic tools to achieve greater accuracy and enable the analysis of large, complex genomes. We apply our pipeline to Zea mays, which has a genome comparable in size to human. Our pipeline begins with the comparison of mass spectra to a putative translation of the genome. We select novel peptides, those that match a region of the genome that was not previously known to be protein coding, for grouping into refinement events. We present a novel, probabilistic framework for evaluating the accuracy of each event. Our calculated event probability, or eventProb, considers the number of supporting peptides and spectra, and the quality of each supporting peptide-spectrum match. Our pipeline predicts 165 novel protein-coding genes and proposes updated models for 741 additional genes.

Integration of omic networks in a developmental atlas of maize

Patterns of development regulation within tissues Expression of a given gene at the RNA level does not always correlate with expression at the protein level for many organisms. Walley et al. have built an integrated atlas of gene expression and regulatory networks in developing maize, using the same tissue samples to measure the transcriptome, proteome, and phosphoproteome. Coexpression networks from the transcriptome and proteome showed little overlap with each other, even though they showed enrichment of similar pathways. Integration of mRNA, protein, and phosphoprotein data sets improved the predictive power of the gene regulatory networks. Science, this issue p. 814 An atlas of the maize functional genome reveals patterns of developmental regulation within tissues for the transcriptome, proteome, and phosphoproteome. Coexpression networks and gene regulatory networks (GRNs) are emerging as important tools for predicting functional roles of individual genes at a system-wide scale. To enable network reconstructions, we built a large-scale gene expression atlas composed of 62,547 messenger RNAs (mRNAs), 17,862 nonmodified proteins, and 6227 phosphoproteins harboring 31,595 phosphorylation sites quantified across maize development. Networks in which nodes are genes connected on the basis of highly correlated expression patterns of mRNAs were very different from networks that were based on coexpression of proteins. Roughly 85% of highly interconnected hubs were not conserved in expression between RNA and protein networks. However, networks from either data type were enriched in similar ontological categories and were effective in predicting known regulatory relationships. Integration of mRNA, protein, and phosphoprotein data sets greatly improved the predictive power of GRNs.

Plastid-produced interorgannellar stress signal MEcPP potentiates induction of the unfolded protein response in endoplasmic reticulum

Significance A defining characteristic of living organisms is dynamic alignment of cellular responses to stress through activation of signal transduction pathways essential for fine-tuning of interorgannellar communication. Uncovering these communication signals is one of the prime challenges of biology. We have identified a chloroplast-produced retrograde signal, methylerythritol cyclodiphosphate (MEcPP), as a trigger of unfolded protein response (UPR) required for restoration of protein-folding homeostasis in the endoplasmic reticulum (ER). Increased levels of MEcPP via genetic manipulation or exogenous application potentiate expression of a sub-set of UPR genes, and alter plant’s resistance to the ER stress inducing agent. These findings provide a link between a plastidial retrograde signal and transcriptional reprogramming of ER genes critical for readjustment of protein-folding capacity in stressed cells. Cellular homeostasis in response to internal and external stimuli requires a tightly coordinated interorgannellar communication network. We recently identified methylerythritol cyclodiphosphate (MEcPP) as a novel stress-specific retrograde signaling metabolite that accumulates in response to environmental perturbations to relay information from plastids to the nucleus. We now demonstrate, using a combination of transcriptome and proteome profiling approaches, that mutant plants (ceh1) with high endogenous levels of MEcPP display increased transcript and protein levels for a subset of the core unfolded protein response (UPR) genes. The UPR is an adaptive cellular response conserved throughout eukaryotes to stress conditions that perturb the endoplasmic reticulum (ER) homeostasis. Our results suggest that MEcPP directly triggers the UPR. Exogenous treatment with MEcPP induces the rapid and transient induction of both the unspliced and spliced forms of the UPR gene bZIP60. Moreover, compared with the parent background (P), ceh1 mutants are less sensitive to the ER-stress-inducing agent tunicamycin (Tm). P and ceh1 plants treated with Tm display similar UPR transcript profiles, suggesting that although MEcPP accumulation causes partial induction of selected UPR genes, full induction is triggered by accumulation of misfolded proteins. This finding refines our perspective of interorgannellar communication by providing a link between a plastidial retrograde signaling molecule and its targeted ensemble of UPR components in ER.

A High-Resolution Tissue-Specific Proteome and Phosphoproteome Atlas of Maize Primary Roots Reveals Functional Gradients along the Root Axes

Tissue-specific protein and phosphoprotein patterns underlie functional gradients along the maize primary root axis. A high-resolution proteome and phosphoproteome atlas of four maize (Zea mays) primary root tissues, the cortex, stele, meristematic zone, and elongation zone, was generated. High-performance liquid chromatography coupled with tandem mass spectrometry identified 11,552 distinct nonmodified and 2,852 phosphorylated proteins across the four root tissues. Two gradients reflecting the abundance of functional protein classes along the longitudinal root axis were observed. While the classes RNA, DNA, and protein peaked in the meristematic zone, cell wall, lipid metabolism, stress, transport, and secondary metabolism culminated in the differentiation zone. Functional specialization of tissues is underscored by six of 10 cortex-specific proteins involved in flavonoid biosynthesis. Comparison of this data set with high-resolution seed and leaf proteome studies revealed 13% (1,504/11,552) root-specific proteins. While only 23% of the 1,504 root-specific proteins accumulated in all four root tissues, 61% of all 11,552 identified proteins accumulated in all four root tissues. This suggests a much higher degree of tissue-specific functionalization of root-specific proteins. In summary, these data illustrate the remarkable plasticity of the proteomic landscape of maize primary roots and thus provide a starting point for gaining a better understanding of their tissue-specific functions.

Dynamic Protein Acetylation in Plant–Pathogen Interactions

Pathogen infection triggers complex molecular perturbations within host cells that results in either resistance or susceptibility. Protein acetylation is an emerging biochemical modification that appears to play central roles during host–pathogen interactions. To date, research in this area has focused on two main themes linking protein acetylation to plant immune signaling. Firstly, it has been established that proper gene expression during defense responses requires modulation of histone acetylation within target gene promoter regions. Second, some pathogens can deliver effector molecules that encode acetyltransferases directly within the host cell to modify acetylation of specific host proteins. Collectively these findings suggest that the acetylation level for a range of host proteins may be modulated to alter the outcome of pathogen infection. This review will focus on summarizing our current understanding of the roles of protein acetylation in plant defense and highlight the utility of proteomics approaches to uncover the complete repertoire of acetylation changes triggered by pathogen infection.

Fungal-induced protein hyperacetylation in maize identified by acetylome profiling

Significance How pathogens manipulate host cellular machinery to enable infection is a major question in biology. The ability of Cochliobolus carbonum race 1 to infect susceptible corn plants relies on production of HC-toxin (HCT). While it is known that HC-toxin is a histone deacetylase inhibitor, knowledge of how HCT actually promotes virulence has remained elusive. Here, we use mass spectrometry to quantify protein abundance and levels of protein acetylation in HCT-treated or pathogen-infected plants. These analyses revealed that the activity of plant-encoded enzymes can be modulated to alter both histone and nonhistone protein acetylation during a susceptible interaction and suggest that virulent C. carbonum utilizes HCT to reprogram the transcriptional response to infection, resulting in an ineffective defense response. Lysine acetylation is a key posttranslational modification that regulates diverse proteins involved in a range of biological processes. The role of histone acetylation in plant defense is well established, and it is known that pathogen effector proteins encoding acetyltransferases can directly acetylate host proteins to alter immunity. However, it is unclear whether endogenous plant enzymes can modulate protein acetylation during an immune response. Here, we investigate how the effector molecule HC-toxin (HCT), a histone deacetylase inhibitor produced by the fungal pathogen Cochliobolus carbonum race 1, promotes virulence in maize through altering protein acetylation. Using mass spectrometry, we globally quantified the abundance of 3,636 proteins and the levels of acetylation at 2,791 sites in maize plants treated with HCT as well as HCT-deficient or HCT-producing strains of C. carbonum. Analyses of these data demonstrate that acetylation is a widespread posttranslational modification impacting proteins encoded by many intensively studied maize genes. Furthermore, the application of exogenous HCT enabled us to show that the activity of plant-encoded enzymes (histone deacetylases) can be modulated to alter acetylation of nonhistone proteins during an immune response. Collectively, these results provide a resource for further mechanistic studies examining the regulation of protein function by reversible acetylation and offer insight into the complex immune response triggered by virulent C. carbonum.

Sample Preparation Protocols for Protein Abundance, Acetylome, and Phosphoproteome Profiling of Plant Tissues

Peptide mass spectrometry is an invaluable technique to globally quantify the proteome. Central to proteome profiling are efficient methods to extract proteins, digest proteins into peptides, and enrich for posttranslationally modified peptides prior to mass spectrometry. In this chapter, we describe methods to extract proteins, process them into peptides, and optionally enrich for phospho- and acetyl-peptides prior to analysis by mass spectrometry.

Dual use of peptide mass spectra: Protein atlas and genome annotation

One of the objectives of genome science is the discovery and accurate annotation of all protein-coding genes. Proteogenomics has emerged as a methodology that provides orthogonal information to traditional forms of evidence used for genome annotation. By this method, peptides that are identified via tandem mass spectrometry are used to refine protein-coding gene models. Namely, these peptides are used to confirm the translation of predicted protein-coding genes, as evidence of novel genes or for correction of current gene models. Proteogenomics requires deep and broad sampling of the proteome in order to generate sufficient numbers of unique peptides. Therefore, we propose that proteogenomic projects are designed so that the generated peptides can also be used to create a comprehensive protein atlas that quantitatively catalogues protein abundance changes during development and in response to environmental stimulus.

Maize defective kernel5 is a bacterial tamB homolog required for chloroplast envelope biogenesis

Chloroplasts are of prokaryotic origin with a double membrane envelope that separates plastid metabolism from the cytosol. Envelope membrane proteins integrate the chloroplast with the cell, but the biogenesis of the envelope membrane remains elusive. We show that the maize defective kernel5 (dek5) locus is critical for plastid membrane biogenesis. Amyloplasts and chloroplasts are larger and reduced in number in dek5 with multiple ultrastructural defects. We show that dek5 encodes a protein homologous to rice SUBSTANDARD STARCH GRAIN4 (SSG4) and E.coli tamB. TamB functions in bacterial outer membrane biogenesis. The DEK5 protein is localized to the chloroplast envelope with a topology analogous to TamB. Increased levels of soluble sugars in dek5 developing endosperm and elevated osmotic pressure in mutant leaf cells suggest defective intracellular solute transport. Both proteomics and antibody-based analyses show that dek5 chloroplasts have reduced levels of chloroplast envelope transporters. Moreover, dek5 chloroplasts reduce inorganic phosphate uptake with at least an 80% reduction relative to normal chloroplasts. These data suggest that DEK5 functions in plastid envelope biogenesis to enable metabolite transport.