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Walley, J. W.

Publications and source records attributed to Walley, J. W..

17 recordsLinked to original sources
bioRxiv2024

Regulatory architecture of disease resistance in maize revealed by multi-omic systems genetics

Complex traits such as disease resistance have been traditionally studied using quantitative genetics. Here, we use systems genetics to integrate disease severity and multi-omic quantitate trait loci (QTL) to uncover biological networks underlying interaction with northern leaf blight (NLB), a yield-limiting disease of corn. Specifically, we integrated transcriptome, (phospho)proteome, and metabolome measurements to map molecular QTL and build predictive regulatory networks following NLB infection. These inferred networks identified a critical signaling module that was genetically validated comprised of a kinase termed NLB SUSCEPTIBLE KINASE 1, a bHLH transcription factor, and the lignin biosynthesis enzyme BROWN MIDRIB 2. Our results demonstrate the feasibility of high-throughput mapping of genetic determinants of gene- product levels and demonstrates the power of systems genetics to identify upstream regulatory genes that confer resistance to NLB that can inform future strategies for crop protection.

plant biology↗
bioRxiv2024

Identification of phenotypic and transcriptomic signatures underpinning maize crown root systems

Maize is pivotal in supporting global agriculture and addressing food security challenges. Crop root systems are critical for water uptake and nutrient acquisition, which impacts yield. Quantitative trait phenotyping is essential to understand better the genetic factors underpinning maize root growth and development. Root systems are challenging to phenotype given their below-ground, soil-bound nature. In addition, manual trait annotations of root images are tedious and can lead to inaccuracies and inconsistencies between individuals, resulting in data discrepancies. To address these issues, we have developed an automated phenotyping pipeline for field-grown maize crown roots by leveraging open-source software. Phenotypic variation of 20 maize genotypes from the Wisconsin Diversity panel was significant for numerous root traits, suggesting a genetic basis for the observed developmental deviations. In addition, juvenile root traits from controlled environment conditions exhibited inconsistent correlation with field-grown adult root traits, underscoring the developmental plasticity prevalent during maize root morphogenesis. Transcripts involved in hormone signaling and stress responses were among differentially expressed genes in roots from 20 maize genotypes, suggesting many molecular processes may underlie the observed phenotypic variance. This study furthers our understanding of genotype-phenotype relationships, which is relevant for informing agricultural strategies to improve maize root physiology.

plant biology↗
bioRxiv2024

Single-cell proteomics differentiates Arabidopsis root cell types

Recently, major advances have enabled the exploration of cellular heterogeneity using single-cell proteomics. Here we examine the feasibility of single-cell proteomics on plant samples. We focus on Arabidopsis thaliana, examining isolated single cells from the cortex and endodermis, which are two adjacent root cell-types derived from a common stem cell. From 756 cells we identify 3,763 proteins and 1,118 proteins/cell. Ultimately, we focus on 3,217 proteins quantified following stringent filtering. Of these, we identified 596 proteins whose expression is enriched in either the cortex or endodermis and are able to differentiate these closely related plant cell-types. Collectivity, our findings underscore the promise of single-cell proteomics to explore the heterogeneity of expression between individual plant cells.

plant biology↗
bioRxiv2023

The Auxin Response Factor ARF27 is required for maize root morphogenesis

Crop root systems are central to nutrition acquisition and water usage. Root hairs and lateral roots contribute to fine-scale patterning of root systems and can offer many advantages for improving root function without drastically impacting overall system architecture. Key genetic regulators underpinning root hair morphogenesis have been well characterized in the model plant Arabidopsis but are less understood in maize. Here, we identify a novel determinant of root hair morphogenesis and auxin responses in maize, AUXIN RESPONSE FACTOR27 (ARF27), using both reverse and quantitative genetic approaches. ARF27 is enriched in maize primary root tissues at both the transcript and protein level. Loss of ARF27 leads to short primary roots and reduced root hair formation, while lateral root density is unaltered. In arf27 roots, auxin-responsive gene expression is dysregulated, which is consistent with the predicted function of this transcription factor. Moreover, a genome wide association study (GWAS) to uncover genetic determinants of auxin-dependent root traits identified ARF27 as a candidate gene. Furthermore, auxin hypersensitive maize genotypes exhibit altered crown root length and surface area in field-grown plants. A gene regulatory network (GRN) was reconstructed and an ARF27 subnetwork was integrated with DAP-seq and GWAS data to identify ARF27 target genes. The ARF27 GRN includes known maize root development genes, such as ROOTLESS CONCERNING CROWN ROOTS (RTCS), ROOTHAIRLESS 3 (RTH3) and RTH6. Altogether this work identifies a novel genetic driver of auxin-mediated root morphogenesis in maize that can inform agricultural strategies for improved crop performance.

plant biology↗
bioRxiv2023

ZmPILS6 is an auxin efflux carrier required for maize root morphogenesis

Plant root systems play a pivotal role in plant physiology and exhibit diverse phenotypic traits. Understanding the genetic mechanisms governing root growth and development in model plants like maize is crucial for enhancing crop resilience to drought and nutrient limitations. This study focused on identifying and characterizing ZmPILS6, an annotated auxin efflux carrier, as a key regulator of various crown root traits in maize. ZmPILS6-modified roots displayed reduced network area and suppressed lateral root formation, desirable traits during drought and low phosphate conditions. The research revealed that ZmPILS6 localizes to the endoplasmic reticulum and plays a vital role in controlling the spatial distribution of indole-3-acetic acid (IAA or "auxin") in primary roots. The study also demonstrated that ZmPILS6 can actively efflux IAA when expressed in yeast. Furthermore, the loss of ZmPILS6 resulted in significant proteome remodeling in maize roots, particularly affecting hormone signaling pathways. To identify potential interacting partners of ZmPILS6, a weighted gene co-expression analysis (WGNA) was performed. Altogether, this research contributes to the growing knowledge of essential genetic determinants governing maize root morphogenesis, which is crucial for guiding agricultural improvement strategies. Significance StatementCrop yield and stress resilience are significantly influenced by crown root architecture. A reverse genetic screen aimed at identifying novel regulators of maize root morphogenesis led to the discovery of ZmPILS6, an auxin efflux carrier. The loss of ZmPILS6 negatively impacts numerous root traits that are linked to plant physiology and function. Proteomic characterization of pils6-1 roots revealed that this evolutionarily conserved transporter is required for the proper expression of numerous phytohormone pathways, including abscisic acid, gibberellins, and jasmonic acid. Notably, ZmPILS6 appears to have a contrasting role in regulating root morphogenesis compared to its Arabidopsis ortholog, PILS6. This finding emphasizes the need for functional characterization of candidate genes directly within key crops of interest, which cannot always be correctly inferred from other model plants.

plant biology↗
bioRxiv2022

Plasmodesmata-located proteins regulate plasmodesmal function at specific cell interfaces in Arabidopsis

Plasmodesmata (PD) are membrane-lined channels connecting adjoining plant cells. PD control symplasmic intercellular communication by allowing molecules to move between cells. Plant polysaccharide callose ({beta}-1,3-glucan) is deposited at PD, affecting plasmodesmal function; however, the regulation of PD at different cell interfaces is largely unknown. This study discovered that two PD-located proteins, PDLP5 and PDLP6, are expressed in non-overlapping cell types. The constitutive expression of PDLP5 and PDLP6 results in the overaccumulation of PD callose at different cell interfaces and starch hyperaccumulation in different cell types within mature leaves. Using a proximity labeling approach, we identified sucrose synthase 6 (SUS6) as a functional partner of PDLP6. We further demonstrated that PDLP6 physically and genetically interacts with SUS6. In addition, callose synthase 7 (CalS7) interacts with both SUS6 and PDLP6 and is required for PDLP6s function. We propose that PDLP6-SUS6-CalS7 forms a callose synthase complex in the vasculature to regulate the plasmodesmal function.

plant biology↗
bioRxiv2022

Temporal and spatial auxin responsive networks in maize primary roots

Auxin is a key regulator of root morphogenesis across angiosperms. To better understand auxin regulated networks underlying maize root development we have characterized auxin responsive transcription across two time points (30 and 120 minutes) and four regions of the primary root: the meristematic zone, elongation zone, cortex, and stele. Hundreds of auxin-regulated genes involved in diverse biological processes were quantified in these different root regions. In general, most auxin regulated genes are region unique and are predominantly observed in differentiated tissues compared to the root meristem. Auxin gene regulatory networks (GRNs) were reconstructed with these data to identify key transcription factors that may underlie auxin responses in maize roots. Additionally, Auxin Response Factor (ARF) subnetworks were generated to identify target genes which exhibit tissue or temporal specificity in response to auxin. These networks describe novel molecular connections underlying maize root development and provide a foundation for functional genomic studies in a key crop.

plant biology↗
bioRxiv2021

slim shady is a novel allele of PHYTOCHROMEB present in the T-DNA line SALK_015201

Auxin is a hormone that is required for hypocotyl elongation during seedling development. In response to auxin rapid changes in transcript and protein abundance occur in hypocotyls and some auxin responsive gene expression is linked to hypocotyl growth. To functionally validate proteomic studies, a reverse genetics screen was performed on mutants in auxin-regulated proteins to identify novel regulators of plant growth. This uncovered a long hypocotyl mutant, which we called slim shady, in an annotated insertion line in IMMUNOREGULATORY RNA-BINDING PROTEIN (IRR). Overexpression of the IRR gene failed to rescue the slim shady phenotype and characterization of a second T-DNA allele of IRR found that it had a wild-type hypocotyl length. The slim shady mutant has an elevated expression of numerous genes associated with the brassinosteroid-auxin-phytochrome (BAP) regulatory module compared to wild-type, including transcription factors that regulate brassinosteroid, auxin and phytochrome pathways. Additionally, slim shady seedlings fail to exhibit a strong transcriptional response to auxin. Using whole genome sequence and transcriptomics data for SALK_015201C we determined that a novel single nucleotide polymorphism in PHYTOCHROME B was responsible for the slim shady phenotype. This is predicted to convert induce a frameshift and premature stop codon at leucine 1125, within the histidine kinase-related domain of the carboxy terminus of PHYB, which is required for phytochrome signaling and function. Genetic complementation analyses with phyb-9 confirmed that slim shady is a mutant allele of PHYB. This study advances our understanding of the molecular mechanisms in seedling development, by furthering our understanding of how light signaling is linked to auxin dependent cell elongation. Furthermore, this study highlights the importance of confirming the genetic identity of research material before attributing phenotypes to known mutations sourced from T-DNA stocks.

plant biology↗
bioRxiv2021

Quantitative proteomics reveals extensive lysine ubiquitination in the Arabidopsis root proteome and uncovers novel transcription factor stability states

Protein activity, abundance, and stability can be regulated by posttranslational modification including ubiquitination. Ubiquitination is conserved among eukaryotes and plays a central role in modulating cellular function and yet we lack comprehensive catalogs of proteins that are modified by ubiquitin in plants. In this study, we describe an antibody-based approach to enrich peptides containing the di-glycine (diGly) remnant of ubiquitin and coupled that with isobaric labeling to enable quantification, from up to 16-multiplexed samples, for plant tissues. Collectively, we identified 7,130 diGly-modified lysine residues sites arising from 3,178 proteins in Arabidopsis primary roots. These data include ubiquitin proteasome dependent ubiquitination events as well as ubiquitination events associated with auxin treatment. Gene Ontology analysis indicated that ubiquitinated proteins are associated with numerous biological processes including hormone signaling, plant defense, protein homeostasis, and root morphogenesis. We determined the ubiquitinated lysine residues that directly regulate the stability of the transcription factors CRYPTOCHROME-INTERACTING BASIC-HELIX-LOOP-HELIX 1 (CIB1), CIB1 LIKE PROTEIN 2 (CIL2), and SENSITIVE TO PROTON RHIZOTOXICITY (STOP1) using site directed mutagenesis and in vivo degradation assays. These comprehensive site-level ubiquitinome profiles provide a wealth of data for future studies related to modulation of biological processes mediated by this posttranslational modification in plants.

plant biology↗
bioRxiv2020

Integrated omics networks reveal the temporal signaling events of brassinosteroid response in Arabidopsis

Brassinosteroids (BRs) are plant steroid hormones that are known to regulate cell division and stress response. We used a systems biology approach to integrate multi-omic datasets and unravel the molecular signaling events of BR response in Arabidopsis. We profiled the levels of 32,549 transcripts, 9,035 protein groups, and 26,950 phosphorylation sites from Arabidopsis seedlings treated with brassinolide (BL, most active BR) for six different lengths of time. We then constructed a network inference pipeline called Spatiotemporal Clustering and Inference of Omics Networks (SC-ION) that was able to integrate these multi-omic data into one, cohesive regulatory network. Our network illustrates the signaling cascade of BR response, starting with kinase-substrate phosphorylation and ending with transcriptional regulation. We used our network predictions to identify putative, relevant phosphorylation sites on the TF BRI1-EMS-SUPPRESSOR 1 (BES1); the importance of which we experimentally validated. Additionally, we identified an uncharacterized TF, which we named BRONTOSAURUS (BRON), that regulates cell division. Further, we show that bron mutant roots are hypersensitive to BL. Together, this work demonstrates the power of integrative network analysis applied to multiomic data and provides fundamental insights into the molecular signaling events occurring during BR response.

plant biology↗
bioRxiv2020

Robotic Assay for Drought (RoAD): An Automated Phenotyping System for Brassinosteroid and Drought Response

Brassinosteroids (BRs) are a group of plant steroid hormones involved in regulating growth, development, and stress responses. Many components of the BR pathway have previously been identified and characterized. However, BR phenotyping experiments are typically performed on petri plates and/or in a low-throughput manner. Additionally, the BR pathway has extensive crosstalk with drought responses, but drought experiments are time-consuming and difficult to control. Thus, we developed Robotic Assay for Drought (RoAD) to perform BR and drought response experiments in soil-grown Arabidopsis plants. RoAD is equipped with a bench scale, a precisely controlled watering system, an RGB camera, and a laser profilometer. It performs daily weighing, watering, and imaging tasks and is capable of administering BR response assays by watering plants with Propiconazole (PCZ), a BR biosynthesis inhibitor. We developed image processing algorithms for both plant segmentation and phenotypic trait extraction in order to accurately measure traits in 2-dimensional (2D) and 3-dimensional (3D) spaces including plant surface area, leaf length, and leaf width. We then applied machine learning algorithms that utilized the extracted phenotypic parameters to identify image-derived traits that can distinguish control, drought, and PCZ-treated plants. We carried out PCZ and drought experiments on a set of BR mutants and Arabidopsis accessions with altered BR responses. Finally, we extended the RoAD assays to perform BR response assays using PCZ in Zea mays (maize) plants. This study establishes an automated and non-invasive robotic imaging system as a tool to accurately measure morphological and growth-related traits of Arabidopsis and maize plants, providing insights into the BR-mediated control of plant growth and stress responses.

plant biology↗
bioRxiv2019

Receptor-Like Kinase Phosphorylation of Arabidopsis Heterotrimeric G-Protein Gα - Subunit AtGPA1

As molecular on-off switches, heterotrimeric G protein complexes, comprised of a G subunit and an obligate G{beta}{gamma} dimer, transmit extracellular signals received by G protein- coupled receptors (GPCRs) to cytoplasmic targets that respond to biotic and abiotic stimuli. Signal transduction is modulated by phosphorylation of GPCRs and G protein complexes. In Arabidopsis thaliana, the G subunit AtGPA1 is phosphorylated by the receptor-like kinase (RLK) BRI1-ASSOCIATED Kinase 1 (BAK1), but the extent that other RLKs phosphorylates AtGPA1 is unknown. We mapped 22 trans-phosphorylation sites on AtGPA1 by 12 RLKs hypothesized to act in the Arabidopsis G protein signaling pathway. Cis-phosphorylation sites on these RLKs were also identified. BRI1, BAK1, and SERK1 have been reported as Ser/Thr and Tyr dual specificity kinases. We identified 4 more dual specificity kinases: IOS1, PSY1R, PEPR1, and AT2G37050. Multiple sites are present in the core AtGPA1 functional units, including pSer52 and pThr53 of the conserved P-loop that directly binds nucleotide/phosphate, pThr164 and pSer175 from E helix in the intramolecular domain interface for nucleotide exchange and GTP hydrolysis, and pThr193 or pThr194 in Switch I (SwI) that coordinates nucleotide exchange and protein partner binding. Several AtGPA1 S/T phosphorylation sites are nucleotide-dependent phosphorylation patterns, such as S52/T53 in the P-loop and T193 and/or T194 in SwI.

plant biology↗
bioRxiv2019

Translational landscape in tomato revealed by transcriptome assembly and ribosome profiling

mRNA translation is a critical step in gene expression, but our understanding of the landscape and control of translation in diverse crops remains lacking. Here, we combined de novo transcriptome assembly and ribosome profiling to study global mRNA translation in tomato roots. Taking advantage of the 3-nucleotide periodicity displayed by translating ribosomes, we identified 354 novel small ORFs (sORFs) translated from previously unannotated transcripts, as well as 1329 upstream ORFs (uORFs) translated within the 5 UTRs of annotated protein-coding genes. Proteomic analysis confirmed that some of these novel uORFs and sORFs generate stable proteins in planta. Compared with the annotated ORFs, the uORFs use more flexible Kozak sequences around translation start sites. Interestingly, uORF-containing genes are enriched for protein phosphorylation/dephosphorylation and signaling transduction pathways, suggesting a regulatory role for uORFs in these processes. We also demonstrated that ribosome profiling is useful to facilitate the annotation of translated ORFs and noncanonical translation initiation sites. In addition to defining the translatome, our results revealed the global control of mRNA translation by uORFs and microRNAs in tomato. In summary, our approach provides a high-throughput method to discover unannotated ORFs, elucidates evolutionarily conserved translational features, and identifies new regulatory mechanisms hidden in a crop genome. SignificanceSeveral studies have shown that altering mRNA translation is a powerful way of improving crop performance. However, due to limited genomic resources and methods, translational regulation remains poorly understood in crops. By leveraging de novo transcriptome assembly and ribosome profiling, we mapped and quantified translating ribosomes across the entire transcriptome in tomato roots. This is the first experiment-based survey to systematically identify actively translated ORFs in a crop. Our results reveal numerous unannotated translation events and uncover new regulatory mechanisms of gene expression in tomato. Our approach not only facilitates our understanding of the tomato translational landscape but also provides a practical strategy to study the translatomes of other species. O_LIThe raw RNA-seq and Ribo-seq data have been deposited in the Gene Expression Omnibus (GEO) database under accession no. GSE124962. C_LIO_LIProteomics raw data files and MaxQuant Search results have been deposited at the MassIVE repository with dataset identifier: MSV000083363. C_LI

plant biology↗
bioRxiv2018

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.

plant biology↗
bioRxiv2018

Assessment and refinement of sample preparation methods for deep and quantitative plant proteome profiling

A major challenge in the field of proteomics is obtaining high quality peptides for comprehensive proteome profiling by liquid chromatography mass spectrometry for many organisms. Here we evaluate and modify a range of sample preparation methods using photosynthetically active Arabidopsis leaf tissues from several developmental timepoints. We find that inclusion of FASP-based on filter digestion improves all protein extraction methods tested. Ultimately, we show that a detergent-free urea-FASP approach enables deep and robust quantification of leaf proteomes. For example, from 4-day-old leaf tissue we profiled up to 11,690 proteins from a single sample replicate. This method should be broadly applicable to researchers working on difficult to process samples from a range of plant and non-plant organisms.\n\nAbbreviations

plant biology↗
bioRxiv2017

Quantitative proteomic analysis of auxin signaling during seedling development

Auxin induces rapid gene expression changes throughout plant development. How these transcriptional responses relate to changes in protein abundance is not well characterized. We have identified auxin regulated proteins in whole seedlings, roots and hypocotyls and at three different time points (30 min, 120 min and 3 hours) using an iTRAQ (isobaric tags for relative and absolute quantification) based quantitative proteomics approach. These profiling experiments detected 4,701 proteins from seedling tissue, 6,740 proteins from root tissue and 3,925 proteins from hypocotyl tissue. Comparisons between the differentially expressed proteins data sets showed little overlap, suggesting that auxin proteomes exhibit both temporal and spatial specificity. Numerous proteins showed significant changes in abundance following auxin treatment independent of changes in cognate transcript abundance. This includes several well characterized proteins with various roles in auxin pathways, suggesting that complex gene regulation mechanisms follow auxin signaling events. Specifically, regulation of translation may play a role as inferred from MapMan categorization analyses and protein interaction networks comprised of auxin regulated proteins. Additionally, functional categorization of auxin regulated proteins indicates rapid and complex metabolic changes occur in both roots and hypocotyls in response to auxin which are not apparent from transcriptome analyses. Altogether these data describe novel auxin-regulated proteomes and are an excellent resource for identifying new downstream signaling components related to auxin-mediated plant growth and development.

plant biology↗