Biology subjects

Shanklin, J.

Publications and source records attributed to Shanklin, J..

13 recordsLinked to original sources

bioRxiv2026

Metabolically engineered oilcane reshapes rhizosphere microbial guilds while preserving broad functional capacity

Metabolic engineering of crops can redirect host carbon flux, but its consequences for microbiomes remain unclear. Here, we show that engineering oilcane for triacylglycerol (TAG) accumulation reshapes rhizosphere microbial guilds across greenhouse and field environments while preserving functional capacity. Using 36 rhizosphere metagenomes from wild-type sugarcane and engineered oilcane accessions, we reconstructed metagenome-assembled genomes and linked community turnover with shifts in functional potential. Oilcane rhizospheres exhibited taxonomic restructuring relative to wild-type plants, driven primarily by turnover rather than nestedness and marked by genotype-dependent replacement of microbial guilds. These patterns were strongest in accession 1566 and amplified under field conditions. Despite these compositional shifts, broad patterns of functional potential remained similarly distributed, whereas pathway-level differences were evident in energy production and conversion, lipid transport and metabolism, secondary metabolite biosynthesis, transport and catabolism, and signal transduction. These findings extend evaluation of engineered crops beyond host traits alone to include microbiome-scale responses.

microbiology↗

bioRxiv2025

Trehalose 6-phosphate activates Target of Rapamycin in plants

Coordinating carbon availability with growth is a fundamental challenge in plants. Target of Rapamycin (TOR) promotes cell growth and is activated by sugars, but the underlying molecular mechanism has remained elusive. Here, we identify trehalose 6-phosphate (T6P), a sucrose-derived metabolite, as the key signal linking carbon to TOR activity. In Arabidopsis and Brassica napus, T6P stimulates cell growth via TOR and is required for sucrose-induced TOR activation. We show immunoprecipitates using anti-TOR antibodies contain catalytically active TOR in addition to the catalytically active SnRK1 energy sensor kinase. In vitro experiments show that, SnRK11, the catalytic subunit of SnRK1 suppresses TOR activity, and T6P reverses this inhibition in a dose-dependent manner, providing biochemical evidence that T6P activates TOR by suppressing SnRK1. This work thus establishes a direct sucrose-T6P-SnRK1-TOR signaling axis that couples carbon availability to plant growth.

plant biology↗

bioRxiv2025

Substrate binding and channeling allosterically modulate the interactions within the AlkB-AlkG electron transfer complex

The transmembrane alkane monooxygenase AlkB and rubredoxin AlkG form an electron transfer complex that hydroxylates terminal alkanes to produce alcohols. The recent cryoEM study of Fontimonas thermophila AlkB-AlkG complex (FtAlkBG) revealed its architecture, including a dodecane (D12) substrate in the diiron active site. However, the molecular mechanism of action of FtAlkBG remains unknown. Here, we examined the FtAlkBG dynamics and interactions by multiscale computations, including molecular dynamics (MD) simulations, elastic network model (ENM), and QM/MM study of the oxidative mechanism at the catalytic site of AlkB. D12 remained stably bound to the catalytic site during MD runs, coordinated by hydrophobic residues I267, L263, L264, and I133, critical to substrate stabilization. MD simulations further showed that D12 could exit the catalytic site via a well-defined translocation pathway gated by I54, S49, and F46, through two intermediate states before nearly leaving the protein, and diffuse back to the active site. Several salt bridges and conserved AlkB-AlkG contacts were enhanced upon substrate binding. Substrate binding also mediated the association/dissociation events required for efficient electron transfer. It promoted tighter coupling between the diiron center in AlkB and the iron in AlkG. The allosteric effects relevant to enzymatic activity regulated by the substrate binding and channeling were further delineated by ENM analysis which confirmed a strong coupling spanning the site of entry from the membrane, the catalytic site and the AlkB-AlkG interface. Our study provides new insights into key sites that could be targeted for developing AlkB-variants with desirable alkane conversion functions.

molecular biology↗

bioRxiv2024

Enhancing lipid production in plant cells through high-throughput genome editing and phenotyping via a scalable automated pipeline

Plant bioengineering is a time-consuming and labor-intensive process, with no guarantee of achieving the desired trait. Here we report a fast, automated, scalable, high-throughput pipeline for plant bioengineering (FAST-PB). FAST-PB achieves gene cloning, genome editing, and product characterization by integrating automated biofoundry engineering of callus and protoplast cells with single cell matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We first demonstrate that FAST-PB can streamline the Golden Gate cloning process, with the capacity to construct 96 vectors in parallel. To prove the concept, using FAST-PB, we first found that PEG2050 significantly increases transfection efficiency by over 45%. To validate the pipeline, we established a reporter-gene-free method for CRISPR editing via mutation of HCF136, affecting cellular chlorophyll fluorescence. Next, we applied this pipeline for lipid production and found that diverse lipids were significantly enhanced up to sixfold through introducing multi-gene cassettes via CRISPR activation, and regenerated plant using this platform. Lastly, we harnessed FAST-PB to achieve high-throughput single-cell lipid profiling through the integration of MALDI-MS with the biofoundry, and differentiated engineered and unengineered cells using the single-cell lipidomics. These innovations massively increase the throughput of synthetic biology, genome editing, and metabolic engineering, and change what is possibly using single-cell metabolomics in plants.

synthetic biology↗

bioRxiv2024

Plasticity of the Arabidopsis leaf lipidome and proteome in response to pathogen infection and heat stress

Plants must cope with a variety of stressors during their life cycle, and the adaptive responses to these environmental cues involve all cellular organelles. Among them, comparatively little is known about the contribution of cytosolic lipid droplets (LDs) and their core set of neutral lipids and associated surface proteins to the rewiring of cellular processes in response to stress. Here, we analyzed the changes that occur in the lipidome and proteome of Arabidopsis leaves after pathogen infection with Botrytis cinerea or Pseudomonas syringae, or after heat stress. Analyses were carried out in wild-type plants and the oil-rich double mutant tgd1-1 sdp1-4 that allowed for an allied study of the LD proteome in stressed leaves. Using liquid chromatography-tandem mass spectrometry-based methods, we show that a hyperaccumulation of the primary LD core lipid triacylglycerol is a general response to stress and that acyl chain and sterol composition are remodeled during cellular adaptation. Likewise, comparative analysis of the LD protein composition in stress-treated leaves highlighted the plasticity of the LD proteome as part of the general stress response. We further identified at least two additional LD-associated proteins, whose localization to LDs in leaves was confirmed by confocal microscopy of fluorescent protein fusions. Taken together, these results highlight LDs as dynamic contributors to the cellular adaptation processes that underlie how plants respond to environmental stress. One sentence summaryBiotic and heat stress strongly alters the lipidome and proteome of Arabidopsis leaves including the proteome of lipid droplets.

plant biology↗

bioRxiv2023

Data-Driven Modeling and Analysis of Fatty Acid Desaturase in Plants

Fatty acids and their derivatives continue to garner attention as sustainable alternatives to petrochemically derived materials. Towards this goal, we examine the plant membrane-bound fatty acid desaturases (FADs) and develop an overview of their 3D structure and phylogenic relationships. Through this effort we developed two plant fatty acid desaturase homology models that we analyzed with substrate docking and molecular dynamics simulations to better understand the diiron binding pocket that is characteristic of these proteins. The comparison between the Omega6 and Delta12 homology models specifically in regards to the binding affinity as a function of carbon chain length indicates that there is a dip in binding affinity near the 18:2 carbon chain length but the rest maintain a rather consistent binding affinity throughout. Furthermore, MD analysis highlights the importance of regions within the cytosolic cap domain on the binding pocket. PACS: 0000, 1111 2000 MSC: 0000, 1111 Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/553759v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@1674c18org.highwire.dtl.DTLVardef@b4e775org.highwire.dtl.DTLVardef@c7ec21org.highwire.dtl.DTLVardef@15fa66c_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDeveloped pipeline to predict function from protein sequence C_LIO_LIModeled two fatty acid desaturases and predicted their structure and functions C_LIO_LILeveraged docking simulations and molecular dynamics to confirm protein function C_LI

molecular biology↗

bioRxiv2022

Genomics of turions from the Greater Duckweed reveal its pathways for dormancy and reemergence strategy

O_LIOver 15 families of aquatic plants are known to use a strategy of developmental switching upon environmental stress to produce dormant propagules called turions. However, few molecular details for turion biology have been elucidated due to the difficulties in isolating high-quality nucleic acids from this tissue. We successfully developed a new protocol to isolate high-quality transcripts and carried out RNA-seq analysis of mature turions from the Greater Duckweed Spirodela polyrhiza. Comparison of turion transcriptome to that of fronds, the actively growing leaf-like tissue, were carried out. C_LIO_LIBioinformatic analysis of high confidence, differentially expressed transcripts between frond and mature turion tissues revealed major pathways related to stress tolerance, starch and lipid metabolism, and dormancy that are mobilized to reprogram frond meristems for turion differentiation. C_LIO_LIWe identified the key genes that are likely to drive starch and lipid accumulation during turion formation, as well as in pathways for starch and lipid utilization upon turion germination. Comparison of genome-wide cytosine methylation levels also revealed evidence for epigenetic changes in the formation of turion tissues. C_LIO_LISimilarities between turions and seeds provided evidence that key regulators for seed maturation and germination have been retooled for their function in turion biology. C_LI

plant biology↗

bioRxiv2021

AlphaFold Protein Structure Database for Sequence-Independent Molecular Replacement

Crystallographic phasing recovers the phase information that is lost during a diffraction experiment. Molecular replacement is a dominant phasing method for the crystal structures in the protein data bank. In one form it uses a protein sequence to search a structure database for finding suitable templates for phasing. However, such sequence information is not always available such as when proteins are crystallized with unknown binding partner proteins or when the crystal is that of a contaminant. The recent development of AlphaFold has resulted in the availability of predicted protein structures for all proteins from twenty species. In this work, we tested whether AlphaFold-predicted E. coli protein structures were accurate enough for sequence-independent phasing of diffraction data from two crystallization contaminants for which we had not identified the protein. Using each of more than 4000 predicted structures as a search model, robust molecular replacement solutions were obtained which allowed the identification and structure determination of both structures, YncE and YadF. Our results advocate a general utility of AlphaFold-predicted structure database with respect to crystallographic phasing.

biochemistry↗

bioRxiv2021

Structural Basis for SARS-CoV-2 Envelope Protein in Recognition of Human Cell Junction Protein PALS1

The COVID-19 pandemic caused by the SARS-CoV-2 virus has created a global health and economic emergency. SARS-CoV-2 viruses hijack human proteins to promote their spread and virulence including the interactions involving the viral envelope (E) protein and human proteins. To understand the structural basis for SARS-CoV-2 viral-host recognition, we used cryo-electron microscopy to determine a structure for the human cell junction protein PALS1 and SARS-CoV-2 E protein complex. The structure shows that the E protein C-terminal DLLV motif recognizes a pocket formed exclusively by hydrophobic residues from the PDZ and SH3 domains in PALS1. Our structural analysis provides an explanation for the observation that the viral E protein recruits PALS1 from lung epithelial cell junctions resulting in vascular leakage, lung damage, viral spread, and virulence. In addition, our structure provides novel targets for peptide- and small-molecule inhibitors that could block the PALS1-E interactions to reduce the E-mediated damage to vascular structures.

biochemistry↗

bioRxiv2020

Genome-wide bioinformatic analyses predict key host and viral factors in SARS-CoV-2 pathogenesis

The novel betacoronavirus named Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) caused a worldwide pandemic (COVID-19) after initially emerging in Wuhan, China. Here we applied a novel, comprehensive bioinformatic strategy to public RNA sequencing and viral genome sequencing data, to better understand how SARS-CoV-2 interacts with human cells. To our knowledge, this is the first meta-analysis to predict host factors that play a specific role in SARS-CoV-2 pathogenesis, distinct from other respiratory viruses. We identified differentially expressed genes, isoforms and transposable element families specifically altered in SARS-CoV-2 infected cells. Well-known immunoregulators including CSF2, IL-32, IL-6 and SERPINA3 were differentially expressed, while immunoregulatory transposable element families were overexpressed. We predicted conserved interactions between the SARS-CoV-2 genome and human RNA-binding proteins such as hnRNPA1, PABPC1 and eIF4b, which may play important roles in the viral life cycle. We also detected four viral sequence variants in the spike, polymerase, and nonstructural proteins that correlate with severity of COVID-19. The host factors we identified likely represent important mechanisms in the disease profile of this pathogen, and could be targeted by prophylactics and/or therapeutics against SARS-CoV-2. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/225581v3_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@15ca5a6org.highwire.dtl.DTLVardef@17f455forg.highwire.dtl.DTLVardef@a39f50org.highwire.dtl.DTLVardef@306ec1_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2020

Structural Basis for Ca2+-Dependent Activation of a Plant Metacaspase

Plants metacaspases mediate programmed cell death in development 1,2, biotic and abiotic stresses 3, damage-induced immune response 4, and resistance to pathogen attack 5. Most metacaspases require Ca2+ for their activation and substrate processing 6-8. However, the Ca2+-dependent activation mechanism remains elusive 9-11. Here we report the crystal structure of Metacaspase 4 from Arabidopsis thaliana (AtMC4) that modulates Ca2+-dependent, damage-induced plant immune defense. The AtMC4 structure exhibits an inhibitory conformation in which a large linker domain blocks activation and substrate access. In addition, the side chain of Lys225 in the linker domain blocks the active site by sitting directly between two catalytic residues. We show that the activation of AtMC4 and cleavage of its physiological substrate involve multiple cleavages in the linker domain upon activation by Ca2+. Our analysis provides insight into the Ca2+-dependent activation of AtMC4 and lays the basis for tuning its activity in response to stresses that may help engineer more sustainable crops for production of food and biofuel.

biochemistry↗

bioRxiv2020

An Expanded Role for WRINKLED1 Metabolic Control Based on Combined Phylogenetic and Biochemical Analyses

During triacylglycerol biosynthesis in developing oilseeds of Arabidopsis thaliana, fatty acid production is regulated by the seed-specific transcription factor WRINKLED1 (WRI1). WRI1 is known to directly stimulate the expression of fatty acid biosynthetic enzymes and a few targets in glycolysis. However, it remains unclear to what extent and how the conversion of sugars into fatty acid biosynthetic precursors in seeds is controlled by WRI1. Based on a previously reported DNA binding motif for WRI1, the ASML1/WRI1 (AW)- box, we developed a comparative genomics approach to search for conserved binding motifs in upstream regions of Arabidopsis thaliana protein-encoding genes and orthologous regions of 11 other Brassicaceae species. The AW-box was over-represented across orthologs for 915 Arabidopsis thaliana genes. Among these, 73 genes with functions in the biosynthesis of fatty acids and triacylglycerols and in glycolysis were enriched. For 90 AW-box sequences associated with these target genes, binding affinity to heterologously expressed Arabidopsis thaliana WRI1 protein was determined using Microscale Thermophoresis. Sites with low dissociation constants are preferentially located close to the transcriptional start site and are highly conserved between the 12 Brassicaceae species. Most of the associated genes were found to be co-expressed with WRI1 during seed development. When 46 automatically and manually curated genes containing conserved AW-sites with high binding affinity are mapped to central metabolism, a conserved regulatory blueprint emerges that infers concerted control of contiguous pathway sections in fatty acid biosynthesis and glycolysis. Among unexpectedly identified putative targets of WRI1 are plastidic fructokinase, phosphoglucose isomerase and several transcription factors. One sentence summaryA combined comparative genomics and in-vitro DNA binding assay approach was used to identify conserved binding sites for the WRINKLED1 transcription factor in central metabolism and lipid biosynthesis.

plant biology↗

bioRxiv2019

BIOTIN ATTACHMENT DOMAIN-CONTAINING proteins, inhibitors of ACCase, are regulated by WRINKLED1

WRINKLED1 (WRI1) is a transcriptional activator that binds to AW boxes in the promoters of many genes from central metabolism and FA synthesis, resulting in their transcription. BIOTIN ATTACHMENT DOMAIN-CONTAINING (BADC) proteins are homologs of BIOTIN CARBOXYL CARRIER PROTEIN (BCCP) that lack a biotin-attachment domain and are therefore inactive. In the presence of excess FA, BADC1 and BADC3 are primarily responsible for the observed long-term irreversible inhibition of ACETYL-COA CARBOXYLASE (ACCase), and consequently FA synthesis. Purified WRI1 bound with high affinity (Kds in the low nanomolar range) to canonical AW-boxes from the promoters of all three BADC genes. Consistent with this observation, the expression of BADC1, BADC2 and BADC3 genes and BADC1 protein levels were reduced in wri1-1 relative to wild type (WT), and BADCs gene expression and BADC1 protein levels also were elevated upon WRI1 overexpression. The double mutant badc1badc2 phenocopied wri1-1 with respect to both reduction in root length, and elevation of indole-3-acetic acid-Asp (IAA-Asp) levels relative to WT. Overexpression of BADC1 in wri1-1 decreased its IAA-Asp and partially rescued its short-root phenotype demonstrating a role for BADCs in seedling establishment. That WRI1 positively regulates genes encoding both FA synthesis and BADCs i.e., conditional inhibitors of FA synthesis, represents a coordinated mechanism to achieve lipid homeostasis in which plants couple the transcription their FA synthetic capacity with their capacity to biochemically downregulate it.\n\nOne sentence summaryWRI1 regulates genes encoding both fatty acid synthesis and inhibitors of FA synthesis (BADCs), creating a lipid homeostatic mechanism in which the transcription of FA synthetic capacity is coordinated with the capacity to biochemically downregulate FA synthesis.

plant biology↗