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All sourcesNASA NTRSDOE OSTI
DOE OSTI2021

The core autophagy machinery is not required for chloroplast singlet oxygen-mediated cell death in the Arabidopsis thaliana plastid ferrochelatase two mutant

Chloroplasts respond to stress and changes in the environment by producing reactive oxygen species (ROS) that have specific signaling abilities. The ROS singlet oxygen ( 1 O 2 ) is unique in that it can signal to initiate cellular degradation including the selective degradation of damaged chloroplasts. This chloroplast quality control pathway can be monitored in the Arabidopsis thaliana mutant plastid ferrochelatase two ( fc2 ) that conditionally accumulates chloroplast 1 O 2 under diurnal light cycling conditions leading to rapid chloroplast degradation and eventual cell death. The cellular machinery involved in such degradation, however, remains unknown. Recently, it was demonstrated that whole damaged chloroplasts can be transported to the central vacuole via a process requiring autophagosomes and core components of the autophagy machinery. The relationship between this process, referred to as chlorophagy, and the degradation of 1 O 2 -stressed chloroplasts and cells has remained unexplored. Results To further understand 1 O 2 -induced cellular degradation and determine what role autophagy may play, the expression of autophagy-related genes was monitored in 1 O 2 -stressed fc2 seedlings and found to be induced. Although autophagosomes were present in fc2 cells, they did not associate with chloroplasts during 1 O 2 stress. Mutations affecting the core autophagy machinery ( atg5 , atg7 , and atg10 ) were unable to suppress 1 O 2 -induced cell death or chloroplast protrusion into the central vacuole, suggesting autophagosome formation is dispensable for such 1 O 2 –mediated cellular degradation. However, both atg5 and atg7 led to specific defects in chloroplast ultrastructure and photosynthetic efficiencies, suggesting core autophagy machinery is involved in protecting chloroplasts from photo-oxidative damage. Finally, genes predicted to be involved in microautophagy were shown to be induced in stressed fc2 seedlings, indicating a possible role for an alternate form of autophagy in the dismantling of 1 O 2 -damaged chloroplasts. Conclusions Our results support the hypothesis that 1 O 2 -dependent cell death is independent from autophagosome formation, canonical autophagy, and chlorophagy. Furthermore, autophagosome-independent microautophagy may be involved in degrading 1 O 2 -damaged chloroplasts. At the same time, canonical autophagy may still play a role in protecting chloroplasts from 1 O 2 -induced photo-oxidative stress. Together, this suggests chloroplast function and degradation is a complex process utilizing multiple autophagy and degradation machineries, possibly depending on the type of stress or damage incurred.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Identification and preliminary characterization of conserved uncharacterized proteins from Chlamydomonas reinhardtii , Arabidopsis thaliana , and Setaria viridis

Abstract The rapid accumulation of sequenced plant genomes in the past decade has outpaced the still difficult problem of genome‐wide protein‐coding gene annotation. A substantial fraction of protein‐coding genes in all plant genomes are poorly annotated or unannotated and remain functionally uncharacterized. We identified unannotated proteins in three model organisms representing distinct branches of the green lineage (Viridiplantae): Arabidopsis thaliana (eudicot), Setaria viridis (monocot), and Chlamydomonas reinhardtii (Chlorophyte alga). Using similarity searching, we identified a subset of unannotated proteins that were conserved between these species and defined them as Deep Green proteins. Bioinformatic, genomic, and structural predictions were performed to begin classifying Deep Green genes and proteins. Compared to whole proteomes for each species, the Deep Green set was enriched for proteins with predicted chloroplast targeting signals predictive of photosynthetic or plastid functions, a result that was consistent with enrichment for daylight phase diurnal expression patterning. Structural predictions using AlphaFold and comparisons to known structures showed that a significant proportion of Deep Green proteins may possess novel folds. Though only available for three organisms, the Deep Green genes and proteins provide a starting resource of high‐value targets for further investigation of potentially new protein structures and functions conserved across the green lineage.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A Sec14 domain protein is required for photoautotrophic growth and chloroplast vesicle formation in Arabidopsis thaliana

In eukaryotic photosynthetic organisms, the conversion of solar into chemical energy occurs in thylakoid membranes in the chloroplast. How thylakoid membranes are formed and maintained is poorly understood. However, previous observations of vesicles adjacent to the stromal side of the inner envelope membrane of the chloroplast suggest a possible role of membrane transport via vesicle trafficking from the inner envelope to the thylakoids. Here we show that the model plant Arabidopsis thaliana has a chloroplast-localized Sec14-like protein (CPSFL1) that is necessary for photoautotrophic growth and vesicle formation at the inner envelope membrane of the chloroplast. The cpsfl1 mutants are seedling lethal, show a defect in thylakoid structure, and lack chloroplast vesicles. Sec14 domain proteins are found only in eukaryotes and have been well characterized in yeast, where they regulate vesicle budding at the trans -Golgi network. Like the yeast Sec14p, CPSFL1 binds phosphatidylinositol phosphates (PIPs) and phosphatidic acid (PA) and acts as a phosphatidylinositol transfer protein in vitro, and expression of Arabidopsis CPSFL1 can complement the yeast sec14 mutation. CPSFL1 can transfer PIP into PA-rich membrane bilayers in vitro, suggesting that CPSFL1 potentially facilitates vesicle formation by trafficking PA and/or PIP, known regulators of membrane trafficking between organellar subcompartments. These results underscore the role of vesicles in thylakoid biogenesis and/or maintenance. CPSFL1 appears to be an example of a eukaryotic cytosolic protein that has been coopted for a function in the chloroplast, an organelle derived from endosymbiosis of a cyanobacterium.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

A phosphorylation-deficient ribosomal protein eS6 is largely functional in Arabidopsis thaliana , rescuing mutant defects from global translation and gene expression to photosynthesis and growth

The eukaryote-specific ribosomal protein of the small subunit eS6 is phosphorylated through the target of rapamycin (TOR) kinase pathway. Although this phosphorylation event responds dynamically to environmental conditions and has been studied for over 50 years, its biochemical and physiological significance remains controversial and poorly understood. Here, we report data from Arabidopsis thaliana, which indicate that plants expressing only a phospho-deficient isoform of eS6 grow essentially normally under laboratory conditions. The eS6z (RPS6A) paralog of eS6 functionally rescued a double mutant in both rps6a and rps6b genes when expressed at approximately twice the wild-type dosage. A mutant isoform of eS6z lacking the major six phosphorylatable serine and threonine residues in its carboxyl-terminal tail also rescued the lethality, rosette growth, and polyribosome loading of the double mutant. This isoform also complemented many mutant phenotypes of rps6 that were newly characterized here, including photosynthetic efficiency, and most of the gene expression defects that were measured by transcriptomics and proteomics. However, compared with plants rescued with a phospho-enabled version of eS6z, the phospho-deficient seedlings retained a mild pointed-leaf phenotype, root growth was reduced, and certain cell cycle-related mRNAs and ribosome biogenesis proteins were misexpressed. The residual defects of the phospho-deficient seedlings could be understood as an incomplete rescue of the rps6 mutant defects. There was little or no evidence for gain-of-function defects. As previously published, the phospho-deficient eS6z also rescued the rps6a and rps6b single mutants; however, phosphorylation of the eS6y (RPS6B) paralog remained lower than predicted, further underscoring that plants can tolerate phospho-deficiency of eS6 well. Our data also yield new insights into how plants cope with mutations in essential, duplicated ribosomal protein isoforms.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Correlated mechanochemical maps of Arabidopsis thaliana primary cell walls using atomic force microscope infrared spectroscopy

Spatial heterogeneity in composition and organisation of the primary cell wall affects the mechanics of cellular morphogenesis. However, directly correlating cell wall composition, organisation and mechanics has been challenging. To overcome this barrier, we applied atomic force microscopy coupled with infrared (AFM-IR) spectroscopy to generate spatially correlated maps of chemical and mechanical properties for paraformaldehyde-fixed, intact Arabidopsis thaliana epidermal cell walls. AFM-IR spectra were deconvoluted by non-negative matrix factorisation (NMF) into a linear combination of IR spectral factors representing sets of chemical groups comprising different cell wall components. This approach enables quantification of chemical composition from IR spectral signatures and visualisation of chemical heterogeneity at nanometer resolution. Cross-correlation analysis of the spatial distribution of NMFs and mechanical properties suggests that the carbohydrate composition of cell wall junctions correlates with increased local stiffness. Together, our work establishes new methodology to use AFM-IR for the mechanochemical analysis of intact plant primary cell walls.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

The extracellular vesicle proteomes of Sorghum bicolor and Arabidopsis thaliana are partially conserved

Abstract Plant extracellular vesicles (EVs) are membrane-bound organelles involved mainly in intercellular communications and defense responses against pathogens. Recent studies have demonstrated the presence of proteins, nucleic acids including small RNAs, and lipids along with other metabolites in plant EVs. Here, we describe the isolation and characterization of EVs from sorghum (Sorghum bicolor). Nanoparticle tracking analysis, dynamic light scattering, and cryo-electron tomography showed the presence of a heterogeneous population of EVs isolated from the apoplastic wash of sorghum leaves. Cryo-electron microscopy revealed that EVs had a median size of 110 nm and distinct populations of vesicles with single or multiple lipid bilayers and low or high amounts of contents. The heterogeneity was further supported by data showing that only a subset of EVs that were stained with a membrane dye, Potomac Gold, were also stained with the membrane-permeant esterase-dependent dye, calcein acetoxymethyl ester. Proteomic analysis identified 437 proteins that were enriched in multiple EV isolations, with the majority of these also found in the EV proteome of Arabidopsis (Arabidopsis thaliana). These data suggest a partial conservation of EV contents and function between the monocot, sorghum, and a distantly related eudicot, Arabidopsis.

36 MATERIALS SCIENCE↗
DOE OSTI2024

A core of cell wall proteins functions in wall integrity responses in Arabidopsis thaliana

Abstract Cell walls surround all plant cells, and their composition and structure are tightly regulated to maintain cellular and organismal homeostasis. In response to wall damage, the cell wall integrity (CWI) system is engaged to ameliorate effects on plant growth. Despite the central role CWI plays in plant development, our current understanding of how this system functions at the molecular level is limited. Here, we investigated the transcriptomes of etiolated seedlings of mutants of Arabidopsis thaliana with defects in three major wall polysaccharides, pectin ( quasimodo2 ), cellulose ( cellulose synthase3 je5 ), and xyloglucan ( xyloglucan xylosyltransferase1 and 2 ), to probe whether changes in the expression of cell wall‐related genes occur and are similar or different when specific wall components are reduced or missing. Many changes occurred in the transcriptomes of pectin‐ and cellulose‐deficient plants, but fewer changes occurred in the transcriptomes of xyloglucan‐deficient plants. We hypothesize that this might be because pectins interact with other wall components and/or integrity sensors, whereas cellulose forms a major load‐bearing component of the wall; defects in either appear to trigger the expression of structural proteins to maintain wall cohesion in the absence of a major polysaccharide. This core set of genes functioning in CWI in plants represents an attractive target for future genetic engineering of robust and resilient cell walls.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Investigating the mechanism of chloroplast singlet oxygen signaling in the Arabidopsis thaliana accelerated cell death 2 mutant

As sessile organisms, plants have evolved complex signaling mechanisms to sense stress and acclimate. This includes the use of reactive oxygen species (ROS) generated during dysfunctional photosynthesis to initiate signaling. One such ROS, singlet oxygen ( 1 O 2 ), can trigger retrograde signaling, chloroplast degradation, and programmed cell death. However, the signaling mechanisms are largely unknown. Several proteins (e.g. PUB4, OXI1, EX1) are proposed to play signaling roles across three Arabidopsis thaliana mutants that conditionally accumulate chloroplast 1 O 2 (fluorescent in blue light (flu), chlorina 1 (ch1), and plastid ferrochelatase 2 (fc2)). We previously demonstrated that these mutants reveal at least two chloroplast 1 O 2 signaling pathways (represented by flu and fc2/ch1). Here, we test if the 1 O 2 -accumulating lesion mimic mutant, accelerated cell death 2 (acd2), also utilizes these pathways. The pub4–6 allele delayed lesion formation in acd2 and restored photosynthetic efficiency and biomass. Conversely, an oxi1 mutation had no measurable effect on these phenotypes. acd2 mutants were not sensitive to excess light (EL) stress, yet pub4–6 and oxi1 both conferred EL tolerance within the acd2 background, suggesting that EL-induced 1 O 2 signaling pathways are independent from spontaneous lesion formation. Thus, 1 O 2 signaling in acd2 may represent a third (partially overlapping) pathway to control cellular degradation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Identification of Metal Stresses in Arabidopsis thaliana Using Hyperspectral Reflectance Imaging

Industrial accidents, such as the Fukushima and Chernobyl disasters, release harmful chemicals into the environment, covering large geographical areas. Natural flora may serve as biological sensors for detecting metal contamination, such as cesium. Spectral detection of plant stresses typically employs a few select wavelengths and often cannot distinguish between different stress phenotypes. In this study, we apply hyperspectral reflectance imaging in the visible and near-infrared along with multivariate curve resolution (MCR) analysis to identify unique spectral signatures of three stresses in Arabidopsis thaliana : salt, copper, and cesium. While all stress conditions result in common stress physiology, hyperspectral reflectance imaging and MCR analysis produced unique spectral signatures that enabled classification of each stress. As the level of potassium was previously shown to affect cesium stress in plants, the response of A. thaliana to cesium stress under variable levels of potassium was also investigated. Increased levels of potassium reduced the spectral response of A. thaliana to cesium and prevented changes to chloroplast cellular organization. While metal stress mechanisms may vary under different environmental conditions, this study demonstrates that hyperspectral reflectance imaging with MCR analysis can distinguish metal stress phenotypes, providing the potential to detect metal contamination across large geographical areas.

multivariate curve resolution↗
DOE OSTI2020

Structure-based enzyme engineering improves donor-substrate recognition of Arabidopsis thaliana glycosyltransferases

Glycosylation of secondary metabolites involves plant UDP-dependent glycosyltransferases (UGTs). UGTs have shown promise as catalysts in the synthesis of glycosides for medical treatment. However, limited understanding at the molecular level due to insufficient biochemical and structural information has hindered potential applications of most of these UGTs. In the absence of experimental crystal structures, we employed advanced molecular modeling and simulations in conjunction with biochemical characterization to design a workflow to study five Group H Arabidopsis thaliana (76E1, 76E2, 76E4, 76E5, 76D1) UGTs. Based on our rational structural manipulation and analysis, we identified key amino acids (P129 in 76D1; D374 in 76E2; K275 in 76E4), which when mutated improved donor substrate recognition than wildtype UGTs. Molecular dynamics simulations and deep learning analysis identified structural differences, which drive substrate preferences. The design of these UGTs with broader substrate specificity may play important role in biotechnological and industrial applications. These findings can also serve as basis to study other plant UGTs and thereby advancing UGT enzyme engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

The cis-regulatory codes of response to combined heat and drought stress in Arabidopsis thaliana

Abstract Plants respond to their environment by dynamically modulating gene expression. A powerful approach for understanding how these responses are regulated is to integrate information about cis-regulatory elements (CREs) into models called cis-regulatory codes. Transcriptional response to combined stress is typically not the sum of the responses to the individual stresses. However, cis-regulatory codes underlying combined stress response have not been established. Here we modeled transcriptional response to single and combined heat and drought stress in Arabidopsis thaliana. We grouped genes by their pattern of response (independent, antagonistic and synergistic) and trained machine learning models to predict their response using putative CREs (pCREs) as features (median F-measure = 0.64). We then developed a deep learning approach to integrate additional omics information (sequence conservation, chromatin accessibility and histone modification) into our models, improving performance by 6.2%. While pCREs important for predicting independent and antagonistic responses tended to resemble binding motifs of transcription factors associated with heat and/or drought stress, important synergistic pCREs resembled binding motifs of transcription factors not known to be associated with stress. These findings demonstrate how in silico approaches can improve our understanding of the complex codes regulating response to combined stress and help us identify prime targets for future characterization.

Azodi, Christina B.↗
DOE OSTI2022

Advanced genomics identifies growth effectors for proteotoxic ER stress recovery in Arabidopsis thaliana

Abstract Adverse environmental and pathophysiological situations can overwhelm the biosynthetic capacity of the endoplasmic reticulum (ER), igniting a potentially lethal condition known as ER stress. ER stress hampers growth and triggers a conserved cytoprotective signaling cascade, the unfolded protein response (UPR) for ER homeostasis. As ER stress subsides, growth is resumed. Despite the pivotal role of the UPR in growth restoration, the underlying mechanisms for growth resumption are yet unknown. To discover these, we undertook a genomics approach in the model plant species Arabidopsis thaliana and mined the gene reprogramming roles of the UPR modulators, basic leucine zipper28 (bZIP28) and bZIP60, in ER stress resolution. Through a network modeling and experimental validation, we identified key genes downstream of the UPR bZIP-transcription factors (bZIP-TFs), and demonstrated their functional roles. Our analyses have set up a critical pipeline for functional gene discovery in ER stress resolution with broad applicability across multicellular eukaryotes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Expression of a mammalian RNA demethylase increases flower number and floral stem branching in Arabidopsis thaliana

Abstract RNA methylation plays a central regulatory role in plant biology and is a relatively new target for plant improvement efforts. In nearly all cases, perturbation of the RNA methylation machinery results in deleterious phenotypes. However, a recent landmark paper reported that transcriptome‐wide use of the human RNA demethylase FTO substantially increased the yield of rice and potatoes. Here, we have performed the first independent replication of those results and demonstrated broader transferability of the trait, finding increased flower and fruit count in the model species Arabidopsis thaliana . We also performed RNA‐seq of our FTO‐transgenic plants, which we analyzed in conjunction with previously published datasets to detect several previously unrecognized patterns in the functional and structural classification of the upregulated and downregulated genes. From these, we present mechanistic hypotheses to explain these surprising results with the goal of spurring more widespread interest in this promising new approach to plant engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Nonpigmented PsbR is involved in the integrity of excitation landscape in higher plant photosystem II, a case study in Arabidopsis thaliana and a mutant

PsbR is a nonpigmented 10 kDa protein in Photosystem II (PSII) in algae and plants. A recent structural study clarified its enigmatic structural location in a Photosystem II megacomplex that has baffled the community for more than four decades. Our current study interrogates whether absence of PsbR affects the overall dynamics of excitation energy migration within light harvesting complexes (LHC) and PSII super assemblies using highly-active PSII membrane particles, so-called BBY particles, isolated from a PsbR deletion mutant (ΔPsbR) of Arabidopsis thaliana. A femto-second (fs)-time-resolved transient absorption experimentation recorded at 77 K with selective excitation of Chl b which is exclusively present in LHCs enabled us to resolve the temporal differences in LHC→LHC and LHC→PSII excitation energy transfer steps. By applying specific target spectro-kinetic models to the transient absorption datasets, we demonstrated that the time constants of Chl a LHC → Chl a LHC excitation transfer significantly elongates in the ΔPsbR LHC-PSII particles, suggestive of the decreased aggregation level of photosynthetic proteins in the mutant. These findings highlight excitation energy transfer integrity in LHC-PSII assembly is not only determined by the pigmented light-harvesting complexes, but also synergistically by the nonpigmented PSII components. Furthermore, the disturbed integrity in dynamics of excitation energy transfer pathway within LHC-PSII supercomplex is discussed in the context of the altered LHC-PSII megacomplexes type I and II architectures which result from the absence of the PsbR protein in higher plant PSII.

Chlorophyll↗
DOE OSTI2024

Nonphotochemical quenching kinetics GWAS in sorghum identifies genes that may play conserved roles in maize and Arabidopsis thaliana photoprotection

SUMMARY Photosynthetic organisms must cope with rapid fluctuations in light intensity. Nonphotochemical quenching (NPQ) enables the dissipation of excess light energy as heat under high light conditions, whereas its relaxation under low light maximizes photosynthetic productivity. We quantified variation in NPQ kinetics across a large sorghum ( Sorghum bicolor ) association panel in four environments, uncovering significant genetic control for NPQ. A genome‐wide association study (GWAS) confidently identified three unique regions in the sorghum genome associated with NPQ and suggestive associations in an additional 61 regions. We detected strong signals from the sorghum ortholog of Arabidopsis thaliana Suppressor Of Variegation 3 ( SVR3 ) involved in plastid–nucleus signaling. By integrating GWAS results for NPQ across maize ( Zea mays ) and sorghum‐association panels, we identified a second gene, Non‐yellowing 1 ( NYE1 ), originally studied by Gregor Mendel in pea ( Pisum sativum ) and involved in the degradation of photosynthetic pigments in light‐harvesting complexes. Analysis of nye1 insertion alleles in A. thaliana confirmed the effect of this gene on NPQ kinetics in eudicots. We extended our comparative genomics GWAS framework across the entire maize and sorghum genomes, identifying four additional loci involved in NPQ kinetics. These results provide a baseline for increasing the accuracy and speed of candidate gene identification for GWAS in species with high linkage disequilibrium.

Plant Sciences↗
DOE OSTI2021

Catalase protects against nonenzymatic decarboxylations during photorespiration in Arabidopsis thaliana

Abstract Photorespiration recovers carbon that would be otherwise lost following the oxygenation reaction of rubisco and production of glycolate. Photorespiration is essential in plants and recycles glycolate into usable metabolic products through reactions spanning the chloroplast, mitochondrion, and peroxisome. Catalase in peroxisomes plays an important role in this process by disproportionating H 2 O 2 resulting from glycolate oxidation into O 2 and water. We hypothesize that catalase in the peroxisome also protects against nonenzymatic decarboxylations between hydrogen peroxide and photorespiratory intermediates (glyoxylate and/or hydroxypyruvate). We test this hypothesis by detailed gas exchange and biochemical analysis of Arabidopsis thaliana mutants lacking peroxisomal catalase. Our results strongly support this hypothesis, with catalase mutants showing gas exchange evidence for an increased stoichiometry of CO 2 release from photorespiration, specifically an increase in the CO 2 compensation point, a photorespiratory‐dependent decrease in the quantum efficiency of CO 2 assimilation, increase in the 12 CO 2 released in a 13 CO 2 background, and an increase in the postillumination CO 2 burst. Further metabolic evidence suggests this excess CO 2 release occurred via the nonenzymatic decarboxylation of hydroxypyruvate. Specifically, the catalase mutant showed an accumulation of photorespiratory intermediates during a transient increase in rubisco oxygenation consistent with this hypothesis. Additionally, end products of alternative hypotheses explaining this excess release were similar between wild type and catalase mutants. Furthermore, the calculated rate of hydroxypyruvate decarboxylation in catalase mutant is much higher than that of glyoxylate decarboxylation. This work provides evidence that these nonenzymatic decarboxylation reactions, predominately hydroxypyruvate decarboxylation, can occur in vivo when photorespiratory metabolism is genetically disrupted.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Deciphering the structure of Arabidopsis thaliana 5-enol-pyruvyl-shikimate-3-phosphate synthase: An essential step toward the discovery of novel inhibitors to supersede glyphosate

Glyphosate interferes with plant aromatic metabolism through the inhibition of 5-enol-pyruvyl-shikimate-3-phosphate (EPSP) synthase [EPSPS, EC 2.5.1.19]. For this reason, EPSPS has been extensively studied in a vast array of organisms. This notwithstanding, up to date, the crystal structure of the protein has been solved exclusively in a few prokaryotes, while that of the plant enzyme has been only deduced in silico by similarity. This study aimed at determining the structure of EPSPS from the plant model species Arabidopsis thaliana, which has been cloned, heterologously expressed and affinity-purified. The kinetic properties of the enzyme have been determined, as well as its susceptibility to the inhibition brought about by glyphosate. The crystal structure of the protein has been resolved at high resolution (1.4 Å), showing open conformation of the enzyme, which is the state ready for substrate/inhibitor binding. This provides a framework for the structure-based design of novel EPSPS inhibitors. Surface regions near the active-site cleft entrance or at the interdomain hinge appear promising for inhibitor selectivity, while bound chloride near the active site is a potential placeholder for anionic moieties of future herbicides.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Predictive Models of Genetic Redundancy in Arabidopsis thaliana

Abstract Genetic redundancy refers to a situation where an individual with a loss-of-function mutation in one gene (single mutant) does not show an apparent phenotype until one or more paralogs are also knocked out (double/higher-order mutant). Previous studies have identified some characteristics common among redundant gene pairs, but a predictive model of genetic redundancy incorporating a wide variety of features derived from accumulating omics and mutant phenotype data is yet to be established. In addition, the relative importance of these features for genetic redundancy remains largely unclear. Here, we establish machine learning models for predicting whether a gene pair is likely redundant or not in the model plant Arabidopsis thaliana based on six feature categories: functional annotations, evolutionary conservation including duplication patterns and mechanisms, epigenetic marks, protein properties including posttranslational modifications, gene expression, and gene network properties. The definition of redundancy, data transformations, feature subsets, and machine learning algorithms used significantly affected model performance based on holdout, testing phenotype data. Among the most important features in predicting gene pairs as redundant were having a paralog(s) from recent duplication events, annotation as a transcription factor, downregulation during stress conditions, and having similar expression patterns under stress conditions. We also explored the potential reasons underlying mispredictions and limitations of our studies. This genetic redundancy model sheds light on characteristics that may contribute to long-term maintenance of paralogs, and will ultimately allow for more targeted generation of functionally informative double mutants, advancing functional genomic studies.

59 BASIC BIOLOGICAL SCIENCES↗