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This study explores the impact of thermal motion on the magnetic compass mechanism in migratory birds, focusing on the radical pair mechanism within cryptochrome photoreceptors. The coherence of radical pairs, crucial for magnetic field inference, is curbed by spin relaxation induced by intra-protein motion. Molecular dynamics simulations, density-functional-theory-based calculations, and spin dynamics calculations were employed, utilizing Bloch-Redfield-Wangsness (BRW) relaxation theory, to investigate compass sensitivity. Previous research hypothesized that European robin's cryptochrome 4a (ErCry4a) optimized intra-protein motion to minimize spin relaxation, enhancing magnetic sensing compared to the plant Arabidopsis thaliana's cryptochrome 1 (AtCry1). Different correlation times of the nuclear hyperfine coupling constants in AtCry1 and ErCry4a were indeed found, leading to distinct radical pair recombination yields in the two species, with ErCry4a showing optimized sensitivity. However, this optimization is likely negligible in realistic spin systems with numerous nuclear spins. Beyond insights in magnetic sensing, the study presents a detailed method employing molecular dynamics simulations to assess for spin relaxation effects on chemical reactions with realistically modelled protein motion, relevant for studying radical pair reactions at finite temperature., (© 2024 The Authors. ChemPhysChem published by Wiley-VCH GmbH.)

Parasitic weeds, such as Orobanche and Striga , threaten crops globally. Contiguous efforts on the discovery and development of structurally novel seed germination stimulants targeting HYPOSENSITIVE TO LIGHT/KARRIKIN INSENSITIVE 2 (HTL/KAI2) have been made with the goal of weed control. Here, we demonstrate that a natural compound dehydrocostus lactone (DCL) exhibits effective "suicide germination" activity against Orobanche cumana and covalently binds to OcKAI2d2 on two catalytic serine sites with the second modification dependent on the first one. The same interactions and covalent modifications of DCL are also confirmed in AtKAI2. Further in-depth evolution analysis indicates that the proposed two catalytic sites are present throughout the streptophyte algae, hornworts, lycophytes, and seed plants. This discovery is particularly noteworthy as it signifies the first confirmation of a plant endogenous molecule directly binding to KAI2, which is valuable for unraveling the elusive identity of the KAI2 ligand and for targeting KAI2 paralogues for the development of novel germination stimulants.

Phytoene desaturase (PDS) is a key rate-limiting enzyme in the carotenoid biosynthesis pathway. Although commercial PDS inhibitors have been developed for decades, it remains necessary to develop novel PDS inhibitors with higher bioactivity. In this work, we used the scaffold hopping and linker modification approaches to design and synthesize a series of compounds ( 7a - 7o , 8a - 8l , and 14a - 14d ). The postemergence application assay demonstrated that 8e and 7e separately showed the best herbicidal activity at 750 g a.i./ha and lower doses (187.5 g, 375g a.i./ha) without no significant toxicity to maize and wheat. The surface plasmon resonance revealed strong binding affinity between 7e and Synechococcus PDS ( Syn PDS). The HPLC analysis confirmed that 8e at 750 g a.i./ha caused significant phytoene accumulation in Arabidopsis seedlings. This work demonstrates the efficacy of structure-guided optimization through scaffold hopping and linker modification to design potent PDS inhibitors with enhanced bioactivity and crop safety.

Long-term visualization of changes in plasma membrane dynamics during important physiological processes can provide intuitive and reliable information in a 4D mode. However, molecular tools that can visualize plasma membranes over extended periods are lacking due to the absence of effective design rules that can specifically track plasma membrane fluorescent dye molecules over time. Using plant plasma membranes as a model, we systematically investigated the effects of different alkyl chain lengths of FMR dye molecules on their performance in imaging plasma membranes. Our findings indicate that alkyl chain length can effectively regulate the permeability of dye molecules across plasma membranes. The study confirms that introducing medium-length alkyl chains improves the ability of dye molecules to target and anchor to plasma membranes, allowing for long-term imaging of plasma membranes. This provides useful design rules for creating dye molecules that enable long-term visualization of plasma membranes. Using the amphiphilic amino-styryl-pyridine fluorescent skeleton, we discovered that the inclusion of short alkyl chains facilitated rapid crossing of the plasma membrane by the dye molecules, resulting in staining of the cell nucleus and indicating improved cell permeability. Conversely, the inclusion of long alkyl chains hindered the crossing of the cell wall by the dye molecules, preventing staining of the cell membrane and demonstrating membrane impermeability to plant cells. The FMR dyes with medium-length alkyl chains rapidly crossed the cell wall, uniformly stained the cell membrane, and anchored to it for a long period without being transmembrane. This allowed for visualization and tracking of the morphological dynamics of the cell plasma membrane during water loss in a 4D mode. This suggests that the introduction of medium-length alkyl chains into amphiphilic fluorescent dyes can transform them from membrane-permeable fluorescent dyes to membrane-staining fluorescent dyes suitable for long-term imaging of the plasma membrane. In addition, we have successfully converted a membrane-impermeable fluorescent dye molecule into a membrane-staining fluorescent dye by introducing medium-length alkyl chains into the molecule. This molecular engineering of dye molecules with alkyl chains to regulate cell permeability provides a simple and effective design rule for long-term visualization of the plasma membrane, and a convenient and feasible means of chemical modification for efficient transmembrane transport of small molecule drugs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Gene expression in Arabidopsis is regulated by more than 1,900 transcription factors (TFs), which have been identified genome-wide by the presence of well-conserved DNA-binding domains. Activator TFs contain activation domains (ADs) that recruit coactivator complexes; however, for nearly all Arabidopsis TFs, we lack knowledge about the presence, location and transcriptional strength of their ADs 1 . To address this gap, here we use a yeast library approach to experimentally identify Arabidopsis ADs on a proteome-wide scale, and find that more than half of the Arabidopsis TFs contain an AD. We annotate 1,553 ADs, the vast majority of which are, to our knowledge, previously unknown. Using the dataset generated, we develop a neural network to accurately predict ADs and to identify sequence features that are necessary to recruit coactivator complexes. We uncover six distinct combinations of sequence features that result in activation activity, providing a framework to interrogate the subfunctionalization of ADs. Furthermore, we identify ADs in the ancient AUXIN RESPONSE FACTOR family of TFs, revealing that AD positioning is conserved in distinct clades. Our findings provide a deep resource for understanding transcriptional activation, a framework for examining function in intrinsically disordered regions and a predictive model of ADs., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Thiols function as antioxidants in food, prolonging shelf life and enhancing flavor. Moreover, thiols are vital biomolecules involved in enzyme activity, cellular signal transduction, and protein folding among critical biological processes. In this paper, the fluorescent probe PYL-NBD was designed and synthesized, which utilized the fluorescent molecule pyrazoline, the lysosome-targeted morpholine moiety, and the sensing moiety NBD. Probe PYL-NBD was tailored for the recognition of biothiols through single-wavelength excitation, yielding distinct fluorescence emission signals: blue for Cys, Hcy, and GSH; green for Cys, Hcy. Probe PYL-NBD exhibited rapid reaction kinetics (<10 min), distinct fluorescence response signals, and low detection limits (15.7 nM for Cys, 14.4 nM for Hcy, and 12.6 nM for GSH). Probe PYL-NBD enabled quantitative determination of Cys content in food samples and L-cysteine capsules. Furthermore, probe PYL-NBD had been successfully applied for confocal imaging with dual-channel detection of biothiols in various biological specimens, including HeLa cells, zebrafish, tumor sections, and Arabidopsis thaliana., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Fluoride ions (F - ) are one of the essential trace elements for the human body and are widely existed in nature. In this study, we present a novel fluorescent probe (YF-SZ-F) designed and synthesized for the specific detection of F - . The probe exhibits high sensitivity, excellent selectivity, and low cytotoxicity, making it a promising tool for biomedical applications. Imaging experiments conducted on zebrafish and Arabidopsis roots demonstrate the probe's remarkable cellular permeability and biocompatibility, laying a solid foundation for its potential biomedical utility. Furthermore, the probe holds potential for practical applications in environmental monitoring and public health through its capability to detect fluoride ions in water samples and via mobile phone software. This multifaceted functionality underscores the broad applicability and significance of the fluorescent probe, not only in scientific research but also in real-world scenarios, contributing to the development of more convenient and precise methods for fluoride detection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cryptochromes are essential flavoproteins for circadian rhythms and avian magnetoreception. Flavin adenine dinucleotide (FAD), a chromophore within cryptochromes, absorbs blue light, initiating electron transfer processes that lead to a biological signaling cascade. A key step in this cascade is the formation of the FAD semiquinone radical (FADH • ), characterized through a specific red-light absorption. The absorption spectra of FADH • in cryptochromes are, however, significantly different from those recorded for the cofactor in solution, primarily due to protein-induced shifts in the absorption peaks. This study employs a multiscale approach, combining molecular dynamics (MD) simulations with quantum mechanical/molecular mechanical (QM/MM) methodologies, to investigate the influence of protein dynamics on embedded FADH • absorption. We emphasize the role of the protein's polarizable environment in the shaping of the absorption spectrum, crucial for accurate spectral predictions in cryptochromes. Our findings provide valuable insights into the absorption process, advancing our understanding of cryptochrome functioning.

The uptake of plastic particles by plants and their transport through the food chain make great risks to biota and human health. Therefore, it is important to trace plastic particles in the plant. Traditional fluorescence imaging in plants usually suffers significant autofluorescence background. Here, we report a persistent luminescence nanoplatform for autofluorescence-free imaging and quantitation of submicrometer plastic particles in plant. The nanoplatform was fabricated by doping persistent luminescence nanoparticles (PLNPs) onto polystyrene (PS) nanoparticles. Cr 3+ -doped zinc gallate PLNP was employed as the dopant for autofluorescence-free imaging due to its persistent luminescence nature. In addition, the Ga element in PLNP was used as a proxy to quantify the PS in the plant by inductively coupled plasma mass spectrometry (ICP-MS). Thus, the developed nanoplatform allows not only dual-mode autofluorescence-free imaging (persistent luminescence and laser-ablation ICP-MS) but also ICP-MS quantitation for tracking PS in plant. Application of this nanoplatform in a typical plant model Arabidopsis thaliana revealed that PS mainly distributed in the root (>99.45%) and translocated very limited (<0.55%) to the shoot. The developed nanoplatform has great potential for quantitative tracing of submicrometer plastic particles to investigate the environmental process and impact of plastic particles.

Background: Cysteine (Cys), glutathione (GSH), and homocysteine (Hcy), as three major biothiols are involved in a variety of physiological processes and play a crucial role in plant growth. Abnormal levels of Cys can cause plants to fail to grow properly. To date, although a very large number of fluorescent probes have been reported for the detection of biothiols, very few of them can be used for the selective discrimination of Cys from GSH and Hcy due to their structural similarity, and only a few of them can be used for plant imaging., Results: Here, three fluorescent probes (o-/m-/p-TMA) based on TMN fluorophore and the ortho-/meta-/para-substituted maleimide recognition groups were constructed to investigate the selective response effect of Cys. Compared to the o-/m-TMA, p-TMA can selectively detect Cys over GSH and Hcy with a rapid response time (10 min) and a low detection limit (0.26 μM). The theoretical calculation confirmed that the intermediate p-TMA-Cys-int has shorter interatomic reaction distances (3.827 Å) compared to o-/m-TMA-Cys (5.533/5.287 Å), making it more suitable for further transcyclization reactions. Additionally, p-TMA has been employed for selective tracking of exogenous and endogenous Cys in Arabidopsis thaliana using both single-/two-photon fluorescence imaging. Furthermore, single cell walls produced obvious two-photon fluorescence signals, indicating that p-TMA can be used for high-concentration Cys analysis in single cells. Surprisingly, p-TMA can be used as a fluorescent dye for protein staining in SDS-PAGE with higher sensitivity (7.49 μg/mL) than classical Coomassie brilliant blue (14.11 μg/mL)., Significance: The outstanding properties of p-TMA make it a promising multifunctional molecular tool for the highly selective detection of Cys over GSH and Hcy in various complex environments, including water solutions, zebrafish, and plants. Additionally, it has the potential to be developed as a fluorescent dye for a simple and fast SDS-PAGE fluorescence staining method., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Natural light harvesting is exceptionally efficient thanks to the local energy funnel created within light-harvesting complexes (LHCs). To understand the design principles underlying energy transport in LHCs, ultrafast spectroscopy is often complemented by mutational studies that introduce perturbations into the excitonic structure of the natural complexes. However, such studies may fall short of identifying all excitation energy transfer (EET) pathways and their changes upon mutation. Here, we show that a synergistic combination of first-principles calculations and ultrafast spectroscopy can give unprecedented insight into the EET pathways occurring within LHCs. We measured the transient absorption spectra of the minor CP29 complex of plants and of two mutants, systematically mapping the kinetic components seen in experiments to the simulated exciton dynamics. With our combined strategy, we show that EET in CP29 is surprisingly robust to the changes in the exciton states induced by mutations, explaining the versatility of plant LHCs.

Mediator complex is a key component that bridges various transcription activators and RNA polymerase during eukaryotic transcription initiation. The Arabidopsis thaliana Med25 (aMed25), a subunit of the Mediator complex, plays important roles in regulating hormone signaling, biotic and abiotic stress responses and plant development by interacting with a variety of transcription factors through its activator-interacting domain (ACID). However, the recognition mechanism of aMed25-ACID for various transcription factors remains unknown. Here, we report the nearly complete 1 H, 13 C, and 15 N backbone and side chain resonance assignments of aMED25-ACID (residues 551-681). TALOS-N analysis revealed that aMED25-ACID structure is comprised of three α-helices and seven β-strands, which lacks the C-terminal α-helix existing in the human MED25-ACID. This study lays a foundation for further research on the structure-function relationship of aMED25-ACID., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Natural rubber produced in stems of the guayule plant (Parthenium argentatum) is susceptible to post-harvest degradation from microbial or thermo-oxidative processes, especially once stems are chipped. As a result, the time from harvest to extraction must be minimized to recover high quality rubber, especially in warm summer months. Tocopherols are natural antioxidants produced in plants through the shikimate and methyl-erythtiol-4-phosphate (MEP) pathways. We hypothesized that increased in vivo guayule tocopherol content might protect rubber from post-harvest degradation, and/or allow reduced use of chemical antioxidants during the extraction process. With the objective of enhancing tocopherol content in guayule, we overexpressed four Arabidopsis thaliana tocopherol pathway genes in AZ-2 guayule via Agrobacterium-mediated transformation. Tocopherol content was increased in leaf and stem tissues of most transgenic lines, and some improvement in thermo-oxidative stability was observed. Overexpression of the four tocopherol biosynthesis enzymes, however, altered other isoprenoid pathways resulting in reduced rubber, resin and argentatins content in guayule stems. The latter molecules are mainly synthesized from precursors derived from the mevalonate (MVA) pathway. Our results suggest the existence of crosstalk between the MEP and MVA pathways in guayule and the possibility that carbon metabolism through the MEP pathway impacts rubber biosynthesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

Flavin-based fluorescent proteins are oxygen-independent reporters that hold great promise for imaging anaerobic and hypoxic biological systems. In this study, we explored the feasibility of applying circular permutation, a valuable method for the creation of fluorescent sensors, to flavin-based fluorescent proteins. We used rational design and structural data to identify a suitable location for circular permutation in iLOV, a flavin-based reporter derived from A. thaliana. However, relocating the N- and C-termini to this position resulted in a significant reduction in fluorescence. This loss of fluorescence was reversible, however, by fusing dimerizing coiled coils at the new N- and C-termini to compensate for the increase in local chain entropy. Additionally, by inserting protease cleavage sites in circularly permuted iLOV, we developed two protease sensors and demonstrated their application in mammalian cells. In summary, our work establishes the first approach to engineer circularly permuted FbFPs optimized for high fluorescence and further showcases the utility of circularly permuted FbFPs to serve as a scaffold for sensor engineering., (© 2024 Wiley-VCH GmbH.)

Intrinsically disordered late embryogenesis abundant (LEA) proteins play a central role in the tolerance of plants and other organisms to dehydration brought upon, for example, by freezing temperatures, high salt concentration, drought or desiccation, and many LEA proteins have been found to stabilize dehydration-sensitive cellular structures. Their conformational ensembles are highly sensitive to the environment, allowing them to undergo conformational changes and adopt ordered secondary and quaternary structures and to participate in formation of membraneless organelles. In an interdisciplinary approach, we discovered how the functional diversity of the Arabidopsis thaliana LEA protein COR15A found in vitro is encoded in its structural repertoire, with the stabilization of membranes being achieved at the level of secondary structure and the stabilization of enzymes accomplished by the formation of oligomeric complexes. We provide molecular details on intra- and inter-monomeric helix-helix interactions, demonstrate how oligomerization is driven by an α-helical molecular recognition feature (α-MoRF) and provide a rationale that the formation of noncanonical, loosely packed, right-handed coiled-coils might be a recurring theme for homo- and hetero-oligomerization of LEA proteins., (© 2024 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

The plant hormone abscisic acid (ABA) plays a critical role in various environmental stress responses and has long been expected to be used in agriculture. However, the practical use of ABA has been limited, mainly because of its photoinstability and rapid biodegradation. We previously developed photostable ABA agonists, BP2A and Me 1',4'- trans -diol BP2A, in which the dienoic acid side chain of ABA was replaced with phenylacetic acid. This finding validated our structure-based approach in designing photostable agonists and provided a basis for developing a more potent or long-lasting ABA agonist. In this study, we synthesized novel BP2A analogs in which the cyclohexenone ring was modified to avoid catabolism by the ABA metabolic enzyme, ABA 8'-hydroxylase. All synthesized analogs showed higher photostability than BP2A under sunlight. In an Arabidopsis seed germination assay, (+)-compounds 5 and 6 with a tetralone ring displayed significantly stronger ABA agonist activity than (+)-BP2A. In contrast, in the in vitro phosphatase assays, both compounds showed comparable or weaker ABA receptor (PYL1) agonistic activity than (+)-BP2A, suggesting that the stronger ABA-like activity of (+)-5 and (+)-6 may arise from their metabolic stability in vivo. This study provides data relevant to designing photostable and persistent ABA agonists.

In plant cells, cellulose synthase complexes (CSCs) are nanoscale machines that synthesize and extrude crystalline cellulose microfibrils (CMFs) into the apoplast where CMFs are assembled with other matrix polymers into specific structures. We report the tissue-specific directionality of CSC movements of the xylem and interfascicular fiber walls of Arabidopsis stems, inferred from the polarity of CMFs determined using vibrational sum frequency generation spectroscopy. CMFs in xylems are deposited in an unidirectionally biased pattern with their alignment axes tilted about 25° off the stem axis, while interfascicular fibers are bidirectional and highly aligned along the longitudinal axis of the stem. These structures are compatible with the design of fiber-reinforced composites for tubular conduit and support pillar, respectively, suggesting that during cell development, CSC movement is regulated to produce wall structures optimized for cell-specific functions., (© 2023. The Author(s).)

Extracellular perception of auxin, an essential phytohormone in plants, has been debated for decades. Auxin-binding protein 1 (ABP1) physically interacts with quintessential transmembrane kinases (TMKs) and was proposed to act as an extracellular auxin receptor, but its role was disputed because abp1 knockout mutants lack obvious morphological phenotypes. Here, we identified two new auxin-binding proteins, ABL1 and ABL2, that are localized to the apoplast and directly interact with the extracellular domain of TMKs in an auxin-dependent manner. Furthermore, functionally redundant ABL1 and ABL2 genetically interact with TMKs and exhibit functions that overlap with those of ABP1 as well as being independent of ABP1. Importantly, the extracellular domain of TMK1 itself binds auxin and synergizes with either ABP1 or ABL1 in auxin binding. Thus, our findings discovered auxin receptors ABL1 and ABL2 having functions overlapping with but distinct from ABP1 and acting together with TMKs as co-receptors for extracellular auxin., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Within the cell, the trace element molybdenum (Mo) is only biologically active when complexed either within the nitrogenase-specific FeMo cofactor or within the molybdenum cofactor (Moco). Moco consists of an organic part, called molybdopterin (MPT) and an inorganic part, that is, the Mo-center. The enzyme which catalyzes the Mo-center formation is the molybdenum insertase (Mo-insertase). Mo-insertases consist of two functional domains called G- and E-domain. The G-domain catalyzes the formation of adenylated MPT (MPT-AMP), which is the substrate for the E-domain, that catalyzes the actual molybdate insertion reaction. Though the functions of E- and G-domain have been elucidated to great structural and mechanistic detail, their combined function is poorly characterized. In this work, we describe a structural model of the eukaryotic Mo-insertase Cnx1 complex that was generated based on cross-linking mass spectrometry combined with computational modeling. We revealed Cnx1 to form an asymmetric hexameric complex which allows the E- and G-domain active sites to align in a catalytic productive orientation toward each other., (© 2023 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Glucosinolates (GSLs) are specialized metabolites in plants of the order Brassicales. GSL transporters (GTRs) are essential for the redistribution of GSLs and also play a role in controlling the GSL content of seeds. However, specific inhibitors of these transporters have not been reported. In the current study, we described the design and synthesis of 2,3,4,6-tetrachloro-5-cyanophenyl GSL (TCPG), an artificial GSL bearing a chlorothalonil moiety as a potent inhibitor of GTRs, and evaluated its inhibitory effect on the substrate uptake mediated through GTR1 and GTR2. Molecular docking showed that the position of the β-D-glucose group of TCPG was significantly different from that of the natural substrate in GTRs and the chlorothalonil moiety forms halogen bonds with GTRs. Functional assays and kinetic analysis of the transport activity revealed that TCPG could significantly inhibit the transport activity of GTR1 and GTR2 (IC 50 values (mean ± SD) being 79 ± 16 μM and 192 ± 14 μM, respectively). Similarly, TCPG could inhibit the uptake and phloem transport of exogenous sinigrin by Arabidopsis thaliana (L.) Heynh leaf tissues, while not affecting that of esculin (a fluorescent surrogate for sucrose). TCPG could also reduce the content of endogenous GSLs in phloem exudates. Together, TCPG was discovered as an undescribed inhibitor of the uptake and phloem transport of GSLs, which brings novel insights into the ligand recognition of GTRs and provides a new strategy to control the GSL level. Further tests on the ecotoxicological and environmental safety of TCPG are needed before using it as an agricultural or horticultural chemical in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Triketones are suitable compounds for 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibition and are important compounds for eliminating agricultural weeds. We report herein quantitative structure-activity relationship (QSAR) modelling and docking studies for a series of triketone-quinoline hybrids and 2-(aryloxyacetyl)cyclohexane-1,3-diones with the aim of proposing new chemical candidates that exhibit improved performance as herbicides. The QSAR models obtained were reliable and predictive (average r 2 , q 2 , and r 2 pred of 0.72, 0.51, and 0.71, respectively). Guided by multivariate image analysis of the PLS regression coefficients and variable importance in projection scores, the substituent effects could be analysed, and a promising derivative with R 1  = H, R 2  = CN, and R 3  = 5,7,8-triCl at the triketone-quinoline scaffold (P18) was proposed. Docking studies demonstrated that π-π stacking interactions and specific interactions between the substituents and amino acid residues in the binding site of the Arabidopsis thaliana HPPD ( At HPPD) enzyme support the desired bioactivity. In addition, compared to a benchmark commercial triketone (mesotrione), the proposed compounds are more lipophilic and less mobile in soil rich in organic matter and are less prone to contaminate groundwater.

Isoprenoids in plants are synthesized following the plastidial methylerythritol-4-phosphate (MEP) pathway or the mevalonate pathway localized to the cytosol and peroxisomes. Isoprenyl-diphosphates (isoprenyl-PP) are important intermediates for the synthesis of chlorophyll, carotenoids, sterols, and other isoprenoids in plants. The quantification of isoprenyl-PP is challenging due to the amphipathic structure, the low abundance, and the susceptibility to hydrolysis during extraction and storage. Different methods for the measurement of isoprenyl-phosphates have been developed. Isoprenyl-phosphates can be measured after radioactive labeling or after derivatization. Liquid chromatography-mass spectrometry (LC-MS) methods provide enhanced sensitivity, but still require the extraction from large amounts of sample material. In the protocol presented here, the monophosphates and diphosphates of farnesol, geranylgeraniol and phytol are isolated from plant material with an isopropanol-containing buffer and quantified by LC-MS using citronellyl-P and citronellyl-PP as internal standards. With a low limit of detection for phytyl-P, geranylgeranyl-P, phytyl-PP, and geranylgeranyl-PP, isoprenyl-phosphates can be accurately measured in Arabidopsis leaves or seeds starting with only 20mg of fresh weight., (Copyright © 2023 Elsevier Inc. All rights reserved.)

α-Helical transmembrane proteins termed membrane transporters mediate the passage of small hydrophilic substrate molecules across biological lipid bilayer membranes. Annotating the specific substrates of the dozens to hundreds of individual transporters of an organism is an important task. In the past, machine learning classifiers have been successfully trained on pan-organism data sets to predict putative substrates of transporters. Here, we critically examine the selection of an optimal data set of protein sequence features for the classification task. We focus on membrane transporters of the three model organisms Escherichia coli , Arabidopsis thaliana , and Saccharomyces cerevisiae , as well as human. We show that organism-specific classifiers can be robustly trained if at least 20 samples are available for each substrate class. If information from position-specific scoring matrices is included, such classifiers have F1 scores between 0.85 and 1.00. For the largest data set ( A. thaliana ), a 4-class classifier yielded an F -score of 0.97. On a pan-organism data set composed of transporters of all four organisms, amino acid and sugar transporters were predicted with an F1 score of 0.91.

The glucosinolate-myrosinase system is a two-component defense system characteristic of cruciferous plants. To evade the glucosinolate-myrosinase system, the crucifer specialist insect, Plutella xylostella , promptly desulfates the glucosinolates into harmless compounds by glucosinolate sulfatases (GSSs) in the gut. In this study, we identified an effective inhibitor of GSSs by virtual screening, molecular docking analysis, and in vitro enzyme inhibition assay. The combined effect of the GSS inhibitor with the plant glucosinolate-myrosinase system was assessed by the bioassay of P. xylostella . We show that irosustat is a GSS inhibitor and the inhibition of GSSs impairs the ability of P. xylostella to detoxify the glucosinolate-myrosinase system, leading to the systematic accumulation of toxic isothiocyanates in larvae, thereby severely affecting feeding, growth, survival, and reproduction of P. xylostella . While fed on the Arabidopsis mutants deficient in myrosinase or glucosinolates, irosustat had no significant negative effect on P. xylostella . These findings reveal that the GSS inhibitor is a novel friendly insecticide to control P. xylostella utilizing the plant glucosinolate-myrosinase system and promote the development of insecticide-plant chemical defense combination strategies.

In the current study, we have utilized semi-enclosed, leak-proof, microfluidic paper-based analytical devices (μPAD's) modified with isatin conjugated chitosan as specific colorimetric reagent for the detection of proline. Proline is one of the globally accepted stress biomarker in plants and also one of the prominent amino acid present in wine and some processed food. Quantification of proline is regularly required in agriculture field, food and wine industries. Specific interaction of isatin with proline, uniform film forming ability of chitosan which results in uniform coloration and the presence of leak-proof layer which prevent the diffusion of colorimetric reagent deeper resulted in enhancement of color signal intensity at the reaction zone were utilized. Further, the images of the μPAD's were captured using smartphone with 3D printed imaging box which houses smartphone and μPAD's. This platform utilizes smartphone flash for uniform illumination and ensures constant positioning of μPAD's to capture images. This greatly enhances the sensitivity and accuracy of our platform. Compared to previously published highly sensitive multi-layer, paper-based platform for detection of proline, current method has enhanced detection range (∼7 fold) and has comparable limit of detection of 23.75 μM. Moreover, the developed μPAD's platform has reduced optimum reaction temperature and time compared to previous work. The developed paper based platform was utilized for evaluation of proline content in young Arabidopsis plants which are subjected to water stress for 5 days. The devised paper-based methods have the potential to be applicable for the on-site evaluation of various stresses in plants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Auxins are hormones that have central roles and control nearly all aspects of growth and development in plants 1-3 . The proteins in the PIN-FORMED (PIN) family (also known as the auxin efflux carrier family) are key participants in this process and control auxin export from the cytosol to the extracellular space 4-9 . Owing to a lack of structural and biochemical data, the molecular mechanism of PIN-mediated auxin transport is not understood. Here we present biophysical analysis together with three structures of Arabidopsis thaliana PIN8: two outward-facing conformations with and without auxin, and one inward-facing conformation bound to the herbicide naphthylphthalamic acid. The structure forms a homodimer, with each monomer divided into a transport and scaffold domain with a clearly defined auxin binding site. Next to the binding site, a proline-proline crossover is a pivot point for structural changes associated with transport, which we show to be independent of proton and ion gradients and probably driven by the negative charge of the auxin. The structures and biochemical data reveal an elevator-type transport mechanism reminiscent of bile acid/sodium symporters, bicarbonate/sodium symporters and sodium/proton antiporters. Our results provide a comprehensive molecular model for auxin recognition and transport by PINs, link and expand on a well-known conceptual framework for transport, and explain a central mechanism of polar auxin transport, a core feature of plant physiology, growth and development., (© 2022. The Author(s).)

The PIN-FORMED (PIN) protein family of auxin transporters mediates polar auxin transport and has crucial roles in plant growth and development 1,2 . Here we present cryo-electron microscopy structures of PIN3 from Arabidopsis thaliana in the apo state and in complex with its substrate indole-3-acetic acid and the inhibitor N-1-naphthylphthalamic acid (NPA). A. thaliana PIN3 exists as a homodimer, and its transmembrane helices 1, 2 and 7 in the scaffold domain are involved in dimerization. The dimeric PIN3 forms a large, joint extracellular-facing cavity at the dimer interface while each subunit adopts an inward-facing conformation. The structural and functional analyses, along with computational studies, reveal the structural basis for the recognition of indole-3-acetic acid and NPA and elucidate the molecular mechanism of NPA inhibition on PIN-mediated auxin transport. The PIN3 structures support an elevator-like model for the transport of auxin, whereby the transport domains undergo up-down rigid-body motions and the dimerized scaffold domains remain static., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Plant ascorbate and glutathione metabolism counteracts oxidative stress mediated, for example, by excess light. In this review, we discuss the properties of immunocytochemistry and transmission electron microscopy, redox-sensitive dyes or probes and bright-field microscopy, confocal microscopy or fluorescence microscopy for the visualization and quantification of glutathione at the cellular or subcellular level in plants and the quantification of glutathione from isolated organelles. In previous studies, we showed that subcellular ascorbate and glutathione levels in Arabidopsis are affected by high light stress. The use of light-emitting diodes (LEDs) is gaining increasing importance in growing indoor crops and ornamental plants. A combination of different LED types allows custom-made combinations of wavelengths and prevents damage related to high photon flux rates. In this review we provide an overview on how different light spectra and light intensities affect glutathione metabolism at the cellular and subcellular levels in plants. Findings obtained in our most recent study demonstrate that both light intensity and spectrum significantly affected glutathione metabolism in wheat at the transcriptional level and caused genotype-specific reactions in the investigated Arabidopsis lines., (© 2022. The Author(s).)

Glucosinolates and camalexin are secondary metabolites that, as phytoanticipins and phytoalexins, play a crucial role in plant defence. The present work proposes an improved analytical method for routine analysis and quantification of glucosinolates and camalexin in brassicaceous small-sized samples by using the very specific desulfation process of glucosinolates analysis and the specificity of fluorescence detection for camalexin analysis. The approach is based on a simultaneous ultrasound-assisted extraction followed by a purification on an anion-exchange column. Final analyses are conducted by HPLC-UV-MS for desulfo-glucosinolates and HPLC coupled to a fluorescence detector (HPLC-FLD) for camalexin. The method is linear for glucosinolates (50-3500 µM) and camalexin (0.025-5 µg.mL -1 ) with an LOD/LOQ of 3.8/12.6 µM and 0.014/0.046 µg.mL -1 respectively. The method demonstrated adequate precision, accuracy and trueness on certified reference rapeseed. A practical application of our approach was conducted on different Brassicaceae genera (Barbarea vulgaris, Brassica nigra, Capsella bursa-pastoris, Cardamine hirsuta, Coincya monensis, Sinapis arvensis, and Sisymbrium officinale) and Arabidopsis thaliana genotypes (Columbia and Wassilewskija). Futhermore, different plant organs (seeds and leaves) were analysed, previously inoculated or not with the pathogenic fungus Alternaria brassicicola., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Dehydrin is an intrinsically disordered protein involved in the cold tolerance of plants. Although dehydrins have been thought to protect biomembranes under cold conditions, the underlying protective mechanism has not been confirmed. Here we report that Arabidopsis dehydrin AtHIRD11 inhibited the aggregation of phospholipid liposomes after freezing and thawing. AtHIRD11 showed significantly greater cryoaggregation-prevention activity than cryoprotective agents such as trehalose, proline, and polyethylene glycols. Amino acid sequence segmentation analysis indicated that the K-segment of AtHIRD11 inhibited the cryoaggregation of phosphatidylcholine (PC) liposomes but other segments did not. This showed that K-segments conserved in all dehydrins were likely to be the cryoprotective sites of dehydrins. Amino acid replacement for a typical K-segment (TypK for short) sequence demonstrated that both hydrophobic and charged amino acids were required for the cryoaggregation-prevention activity of PC liposomes. The amino acid shuffling of TypK remarkably reduced cryoprotective activity. Although TypK did not bind to PC liposomes in solution, the addition of liposomes reduced its disordered content under crowded conditions. Together, these results suggested that dehydrins protected biomembranes via conserved K-segments whose sequences were optimized for cryoprotective activities., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Although several large-scale single-cell RNA sequencing (scRNAseq) studies addressing the root of Arabidopsis (Arabidopsis thaliana) have been published, there is still need for a de novo reference map for both root and especially above-ground cell types. As the plants' transcriptome substantially changes throughout the day, shaped by the circadian clock, we performed scRNAseq on both Arabidopsis root and above-ground tissues at defined times of the day. For the root scRNAseq analysis, we used tissue-specific reporter lines grown on plates and harvested at the end of the day (ED). In addition, we submitted above-ground tissues from plants grown on soil at ED and end of the night to scRNAseq, which allowed us to identify common cell types/markers between root and shoot and uncover transcriptome changes to above-ground tissues depending on the time of the day. The dataset was also exploited beyond the traditional scRNAseq analysis to investigate non-annotated and di-cistronic transcripts. We experimentally confirmed the predicted presence of some of these transcripts and also addressed the potential function of a previously unidentified marker gene for dividing cells. In summary, this work provides insights into the spatial control of gene expression from nearly 70,000 cells of Arabidopsis for below- and whole above-ground tissue at single-cell resolution at defined time points., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Lipopolysaccharides, the major outer membrane components of Gram-negative bacteria, are crucial actors of the host-microbial dialogue. They can contribute to the establishment of either symbiosis or bacterial virulence, depending on the bacterial lifestyle. Plant microbiota shows great complexity, promotes plant health and growth and assures protection from pathogens. How plants perceive LPS from plant-associated bacteria and discriminate between beneficial and pathogenic microbes is an open and urgent question. Here, we report on the structure, conformation, membrane properties and immune recognition of LPS isolated from the Arabidopsis thaliana root microbiota member Herbaspirillum sp. Root189. The LPS consists of an O-methylated and variously acetylated D-rhamnose containing polysaccharide with a rather hydrophobic surface. Plant immunology studies in A. thaliana demonstrate that the native acetylated O-antigen shields the LPS from immune recognition whereas the O-deacylated one does not. These findings highlight the role of Herbaspirillum LPS within plant-microbial crosstalk, and how O-antigen modifications influence membrane properties and modulate LPS host recognition., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

In Arabidopsis , vacuolar sorting receptor isoform 1 (VSR1) sorts 12S globulins to the protein storage vacuoles during seed development. Vacuolar sorting is mediated by specific protein-protein interactions between VSR1 and the vacuolar sorting determinant located at the C terminus (ctVSD) on the cargo proteins. Here, we determined the crystal structure of the protease-associated domain of VSR1 (VSR1-PA) in complex with the C-terminal pentapeptide ( 468 RVAAA 472 ) of cruciferin 1, an isoform of 12S globulins. The 468 RVA 470 motif forms a parallel β-sheet with the switch III residues ( 127 TMD 129 ) of VSR1-PA, and the 471 AA 472 motif docks to a cradle formed by the cargo-binding loop ( 95 RGDCYF 100 ), making a hydrophobic interaction with Tyr99. The C-terminal carboxyl group of the ctVSD is recognized by forming salt bridges with Arg95. The C-terminal sequences of cruciferin 1 and vicilin-like storage protein 22 were sufficient to redirect the secretory red fluorescent protein (spRFP) to the vacuoles in Arabidopsis protoplasts. Adding a proline residue to the C terminus of the ctVSD and R95M substitution of VSR1 disrupted receptor-cargo interactions in vitro and led to increased secretion of spRFP in Arabidopsis protoplasts. How VSR1-PA recognizes ctVSDs of other storage proteins was modeled. The last three residues of ctVSD prefer hydrophobic residues because they form a hydrophobic cluster with Tyr99 of VSR1-PA. Due to charge-charge interactions, conserved acidic residues, Asp129 and Glu132, around the cargo-binding site should prefer basic residues over acidic ones in the ctVSD. The structural insights gained may be useful in targeting recombinant proteins to the protein storage vacuoles in seeds., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Thioredoxin (Trx) is a protein that mediates the reducing power transfer from the photosynthetic electron transport system to target enzymes in chloroplasts and regulates their activities. Redox regulation governed by Trx is a system that is central to the adaptation of various chloroplast functions to the ever-changing light environment. However, the factors involved in the opposite reaction (i.e., the oxidation of various enzymes) have yet to be revealed. Recently, it has been suggested that Trx and Trx-like proteins could oxidize Trx-targeted proteins in vitro. To elucidate the in vivo function of these proteins as oxidation factors, we generated mutant plant lines deficient in Trx or Trx-like proteins and studied how the proteins are involved in oxidative regulation in chloroplasts. We found that f -type Trx and two types of Trx-like proteins, Trx-like 2 and atypical Cys His-rich Trx (ACHT), seemed to serve as oxidation factors for Trx-targeted proteins, such as fructose-1,6-bisphosphatase, Rubisco activase, and the γ-subunit of ATP synthase. In addition, ACHT was found to be involved in regulating nonphotochemical quenching, which is the mechanism underlying the thermal dissipation of excess light energy. Overall, these results indicate that Trx and Trx-like proteins regulate chloroplast functions in concert by controlling the redox state of various photosynthesis-related proteins in vivo., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Background: Agriculture faces significant global challenges including climate change and an increasing food demand due to a growing population. Addressing these challenges will require the adoption of transformative innovations into biotechnology practice, such as nanotechnology. Recently, nanomaterials have emerged as unmatched tools for their use as biosensors, or as biomolecule delivery vehicles. Despite their increasingly prolific use, plant-nanomaterial interactions remain poorly characterized, drawing into question the breadth of their utility and their broader environmental compatibility., Results: Herein, we characterize the response of Arabidopsis thaliana to single walled carbon nanotube (SWNT) exposure with two different surface chemistries commonly used for biosensing and nucleic acid delivery: oligonucleotide adsorbed-pristine SWNTs, and polyethyleneimine-SWNTs loaded with plasmid DNA (PEI-SWNTs), both introduced by leaf infiltration. We observed that pristine SWNTs elicit a mild stress response almost undistinguishable from the infiltration process, indicating that these nanomaterials are well-tolerated by the plant. However, PEI-SWNTs induce a much larger transcriptional reprogramming that involves stress, immunity, and senescence responses. PEI-SWNT-induced transcriptional profile is very similar to that of mutant plants displaying a constitutive immune response or treated with stress-priming agrochemicals. We selected molecular markers from our transcriptomic analysis and identified PEI as the main cause of this adverse reaction. We show that PEI-SWNT response is concentration-dependent and, when persistent over time, leads to cell death. We probed a panel of PEI variant-functionalized SWNTs across two plant species and identified biocompatible SWNT surface functionalizations., Conclusions: While SWNTs themselves are well tolerated by plants, SWNTs surface-functionalized with positively charged polymers become toxic and produce cell death. We use molecular markers to identify more biocompatible SWNT formulations. Our results highlight the importance of nanoparticle surface chemistry on their biocompatibility and will facilitate the use of functionalized nanomaterials for agricultural improvement., (© 2021. The Author(s).)

Phosphate, a key plant nutrient, is perceived through inositol polyphosphates (InsPs) by SPX domain-containing proteins. SPX1 an inhibit the PHR2 transcription factor to maintain Pi homeostasis. How SPX1 recognizes an InsP molecule and represses transcription activation by PHR2 remains unclear. Here we show that, upon binding InsP 6 , SPX1 can disrupt PHR2 dimers and form a 1:1 SPX1-PHR2 complex. The complex structure reveals that SPX1 helix α1 can impose a steric hindrance when interacting with the PHR2 dimer. By stabilizing helix α1, InsP 6 allosterically decouples the PHR2 dimer and stabilizes the SPX1-PHR2 interaction. In doing so, InsP 6 further allows SPX1 to engage with the PHR2 MYB domain and sterically block its interaction with DNA. Taken together, our results suggest that, upon sensing the surrogate signals of phosphate, SPX1 inhibits PHR2 via a dual mechanism that attenuates dimerization and DNA binding activities of PHR2., (© 2021. The Author(s).)

Plant acidic peptide: N-glycanase (aPNGase) release N-glycans from glycopeptides during the degradation process of glycoproteins in developing or growing plants. We have previously developed a new method to detect the aPNGase activity in crude extracts, which is prerequisite for the construction of aPNGase knockout or overexpression lines. However, this method has the disadvantage of requiring de-sialylation treatment and a lectin chromatography. In this study, therefore, we improved the simple and accurate method for detecting aPNGase activity using anion-exchange HPLC requiring neither the desialylation treatment nor the lectin affinity chromatography., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In this study, we show that infrared laser ablation atmospheric pressure photoionization mass spectrometry (LAAPPI-MS) imaging with 70 μm lateral resolution allows for the analysis of Arabidopsis thaliana ( A. thaliana ) leaf substructures ranging from single-cell trichomes and the interveinal leaf lamina to primary, secondary, and tertiary veins. The method also showed its potential for depth profiling analysis for the first time by mapping analytes at the different depths of the leaf and spatially resolving the topmost trichomes and cuticular wax layer from the underlying tissues. Negative ion LAAPPI-MS detected many different flavonol glycosides, fatty acids, fatty acid esters, galactolipids, and glycosphingolipids, whose distributions varied significantly between the different substructures of A. thaliana leaves. The results show that LAAPPI-MS provides a highly promising new tool to study the role of metabolites in plants.

In eukaryotes, small RNAs (sRNAs) play critical roles in multiple biological processes. Dicer endonucleases are a central part of sRNA biogenesis. In plants, DICER-LIKE PROTEIN 3 (DCL3) produces 24-nucleotide (nt) small interfering RNAs (siRNAs) that determine the specificity of the RNA-directed DNA methylation pathway. Here, we determined the structure of a DCL3–pre-siRNA complex in an active dicing-competent state. The 5′-phosphorylated A1 of the guide strand and the 1-nt 3′ overhang of the complementary strand are specifically recognized by a positively charged pocket and an aromatic cap, respectively. The 24-nt siRNA length dependence relies on the separation between the 5′-phosphorylated end of the guide RNA and dual cleavage sites formed by the paired ribonuclease III domains. These structural studies, complemented by functional data, provide insight into the dicing principle for Dicers in general.

While the capsid of viruses in the Alphachrysovirus genus is built of subunits of a single coat protein, the capsid of viruses grouped in the Betachrysovirus genus may consist of subunits of two different proteins. For four of these betachrysoviruses, the detected molecular weights of the putative coat proteins differ from the sizes deduced from the nucleic acid sequence. The origin of these modifications remained unclear and it was hypothesized that the coat proteins undergo unspecific degradation. In our study, we show that these modifications are based on processing steps performed by unknown factors present in extracts of several eukaryotic organisms. Furthermore, we show that the C-terminal domain of P3 is fully degraded after capsid processing and particle assembly., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Transgenic Arabidopsis thaliana expressing an anti-rabies monoclonal antibody (mAb), SO57, was obtained using Agrobacterium -mediated floral dip transformation. The endoplasmic reticulum (ER) retention signal Lys-Asp-Glu-Leu (KDEL) was tagged to the C-terminus of the anti-rabies mAb heavy chain to localize the mAb to the ER and enhance its accumulation. When the inaccurately folded proteins accumulated in the ER exceed its storage capacity, it results in stress that can affect plant development and growth. We generated T 1 transformants and obtained homozygous T 3 seeds from transgenic Arabidopsis to investigate the effect of KDEL on plant growth. The germination rate did not significantly differ between plants expressing mAb SO57 without KDEL (SO plant) and mAb SO57 with KDEL (SOK plant). The primary roots of SOK agar media grown plants were slightly shorter than those of SO plants. Transcriptomic analysis showed that expression of all 11 ER stress-related genes were not significantly changed in SOK plants relative to SO plants. SOK plants showed approximately three-fold higher mAb expression levels than those of SO plants. Consequently, the purified mAb amount per unit of SOK plant biomass was approximately three times higher than that of SO plants. A neutralization assay revealed that both plants exhibited efficient rapid fluorescent focus inhibition test values against the rabies virus relative to commercially available human rabies immunoglobulins. KDEL did not upregulate ER stress-related genes; therefore, the enhanced production of the mAb did not affect plant growth. Thus, KDEL fusion is recommended for enhancing mAb production in plant systems.

A significant number of proteins possess sizable intrinsically disordered regions (IDRs). Due to the dynamic nature of IDRs, NMR spectroscopy is often the tool of choice for characterizing these segments. However, the application of NMR to IDRs is often hindered by their instability, spectral overlap and resonance assignment difficulties. Notably, these challenges increase considerably with the size of the IDR. In response to these issues, here we report the use of sortase-mediated ligation (SML) for segmental isotopic labeling of IDR-containing samples. Specifically, we have developed a ligation strategy involving a key segment of the large IDR and adjacent folded headpiece domain comprising the C-terminus of A. thaliana villin 4 (AtVLN4). This procedure significantly reduces the complexity of NMR spectra and enables group identification of signals arising from the labeled IDR fragment, a process we refer to as segmental assignment. The validity of our segmental assignment approach is corroborated by backbone residue-specific assignment of the IDR using a minimal set of standard heteronuclear NMR methods. Using segmental assignment, we further demonstrate that the IDR region adjacent to the headpiece exhibits nonuniform spectral alterations in response to temperature. Subsequent residue-specific characterization revealed two segments within the IDR that responded to temperature in markedly different ways. Overall, this study represents an important step toward the selective labeling and probing of target segments within much larger IDR contexts. Additionally, the approach described offers significant savings in NMR recording time, a valuable advantage for the study of unstable IDRs, their binding interfaces, and functional mechanisms., Competing Interests: The authors have declared that no competing interests exist.

NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) is a key component of the auxin-dependent plant phototropic growth response. We report that NPH3 directly binds polyacidic phospholipids, required for plasma membrane association in darkness. We further demonstrate that blue light induces an immediate phosphorylation of a C-terminal 14-3-3 binding motif in NPH3. Subsequent association of 14-3-3 proteins is causal for the light-induced release of NPH3 from the membrane and accompanied by NPH3 dephosphorylation. In the cytosol, NPH3 dynamically transitions into membraneless condensate-like structures. The dephosphorylated state of the 14-3-3 binding site and NPH3 membrane recruitment are recoverable in darkness. NPH3 variants that constitutively localize either to the membrane or to condensates are non-functional, revealing a fundamental role of the 14-3-3 mediated dynamic change in NPH3 localization for auxin-dependent phototropism. This regulatory mechanism might be of general nature, given that several members of the NPH3-like family interact with 14-3-3 via a C-terminal motif., (© 2021. The Author(s).)

Mechanosensitive (MS) ion channels respond to mechanical stress and convert it into intracellular electric and ionic signals. Five MS channel families have been identified in plants, including the Mid1-Complementing Activity (MCA) channel; however, its activation mechanisms have not been elucidated in detail. We herein demonstrate that the MCA2 channel is a Ca 2+ -permeable MS channel that is directly activated by membrane tension. The N-terminal 173 residues of MCA1 and MCA2 were synthesized in vitro, purified, and reconstituted into artificial liposomal membranes. Liposomes reconstituted with MCA1(1-173) or MCA2(1-173) mediate Ca 2+ influx and the application of pressure to the membrane reconstituted with MCA2(1-173) elicits channel currents. This channel is also activated by voltage. Blockers for MS channels inhibit activation by stretch, but not by voltage. Since MCA proteins are found exclusively in plants, these results suggest that MCA represent plant-specific MS channels that open directly with membrane tension., (© 2021. The Author(s).)

Plant cell walls contain cellulose embedded in matrix polysaccharides. Understanding carbohydrate structures and interactions is critical to the production of biofuel and biomaterials using these natural resources. Here we present a solid-state NMR study of cellulose and pectin in 13 C-labeled cell walls of Arabidopsis wild-type and mutant plants. Using 1D 13 C and 2D 13 C- 13 C correlation experiments, we detected a highly branched arabinan structure in qua2 and tsd2 samples, two allelic mutants for a pectin methyltransferase. Both mutants show close physical association between cellulose and the backbones of pectic homogalacturonan and rhamnogalacturonan-I. Relaxation and dipolar order parameters revealed enhanced microsecond dynamics due to polymer disorder in the mutants, but restricted motional amplitudes due to tighter pectin-cellulose associations. These molecular data shed light on polymer structure and packing in these two pectin mutants, helping to elucidate how pectin could influence cell wall architecture at the nanoscale, cell wall mechanics, and plant growth., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

We describe sample preparation and visualization of fluorescently tagged cellulose synthases in cellulose synthase complexes at the plasma membrane of Arabidopsis hypocotyl epidermal cells using live-cell imaging via spinning disk microscopy. We present a technique for sample mounting that may be suitable for imaging other samples. Additionally, we offer free, open-source solutions for image analysis and provide extensive troubleshooting suggestions. For complete information on the use and execution of this protocol, please refer to McFarlane et al., 2021., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Shoot meristems contain stem cells, and they sustain growth and development of the above-ground tissues in land plants. The HAIRY MERISTEM ( HAM ) family genes, encoding GRAS-domain transcriptional regulators, play essential roles in the control of shoot meristem development and stem cell homeostasis in several flowering plants. Similar to other GRAS proteins, the C-terminal regions of HAM family proteins across land plants are conserved, containing signature motifs that define the GRAS domain. In contrast, the N-terminal regions of HAM family proteins display substantial divergence in sequence and length. Whether the variable and divergent N-termini are required for the conserved functions of HAM proteins is unknown. Our recent work showed that CrHAM - the HAM homolog in the fern Ceratopteris richardii was able to replace the role of type-II HAM genes in Arabidopsis, maintaining established shoot apical meristems and promoting the initiation of new stem cell niches. Here, we provide additional information and show that CrHAM contains a much longer N-terminal region compared to Arabidopsis HAM proteins, which is conserved among different fern HAM homologs. The deletion of this region largely compromises the ability of CrHAM to replace the function of Arabidopsis HAM proteins in shoot meristems. These new data together with previous results suggest that, although lacking the sequence conservation among HAM homologs from different plant lineages, the N-termini are important for the conserved functions of HAM family proteins.

The chlorophyll biosynthesis regulator GENOMES UNCOUPLED 4 (GUN4) is conserved in nearly all oxygenic photosynthetic organisms. Recently, GUN4 has been found to be able to bind the linear tetrapyrroles (bilins) and stimulate the magnesium chelatase activity in the unicellular green alga Chlamydomonas reinhardtii. Here, we characterize GUN4 proteins from Arabidopsis thaliana and the cyanobacterium Synechocystis sp. PCC 6803 for their ability to bind bilins, and present the crystal structures of Synechocystis GUN4 in biliverdin-bound, phycocyanobilin-bound, and phytochromobilin-bound forms at the resolutions of 1.05, 1.10, and 1.70 Å, respectively. These linear molecules adopt a cyclic-helical conformation, and bind more tightly than planar porphyrins to the tetrapyrrole-binding pocket of GUN4. Based on structural comparison, we propose a working model of GUN4 in regulation of tetrapyrrole biosynthetic pathway, and address the role of the bilin-bound GUN4 in retrograde signaling., (© 2021 The Protein Society.)

We have previously demonstrated that Arabidopsis (Arabidopsis thaliana) phloem protein PP2-A1 is an integral component of resistance to the green peach aphid (Myzus persicae). Here, we report that M. persicae overcomes the resistance of PP2-A1 by using the salivary protein Mp1 as an energetic effector and an interactor of AtPP2-A1. Using the RNA interference technique, we demonstrated that Mp1 plays an essential role in the phloem-feeding activity of M. persicae. When the Mp1 gene was silenced, aphids incurred serious impairments not only in phloem-feeding activity, but also in survival and fertility. In essence, phloem-feeding activity was attributed to the molecular interaction between Mp1 and AtPP2-A1. The Mp1 and AtPP2-A1 interactions were localized to plant cell membranes by co-immunoprecipitation and bimolecular fluorescence complementation experiments. Furthermore, the interaction was found to be required for aphid feeding on Arabidopsis phloem. Overall, our results suggest that Mp1 is an important effector of M. persicae and interacts with AtPP2-A1 to facilitate infestation in the plant tissue by this insect., Competing Interests: Declaration of competing interest None declared., (Copyright © 2021 Elsevier Inc. All rights reserved.)