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As global temperatures rise, improving crop yields will require enhancing the thermotolerance of crops. One approach for improving thermotolerance is using bioengineering to increase the thermostability of enzymes catalysing essential biological processes. Photorespiration is an essential recycling process in plants that is integral to photosynthesis and crop growth. The enzymes of photorespiration are targets for enhancing plant thermotolerance as this pathway limits carbon fixation at elevated temperatures. We explored the effects of temperature on the activity of the photorespiratory enzyme glycerate kinase (GLYK) from various organisms and the homologue from the thermophilic alga Cyanidioschyzon merolae was more thermotolerant than those from mesophilic plants, including Arabidopsis thaliana. To understand enzyme features underlying the thermotolerance of C. merolae GLYK (CmGLYK), we performed molecular dynamics simulations using AlphaFold-predicted structures, which revealed greater movement of loop regions of mesophilic plant GLYKs at higher temperatures compared to CmGLYK. Based on these simulations, hybrid proteins were produced and analysed. These hybrid enzymes contained loop regions from CmGLYK replacing the most mobile corresponding loops of AtGLYK. Two of these hybrid enzymes had enhanced thermostability, with melting temperatures increased by 6 °C. One hybrid with three grafted loops maintained higher activity at elevated temperatures. Whilst this hybrid enzyme exhibited enhanced thermostability and a similar K m for ATP compared to AtGLYK, its K m for glycerate increased threefold. This study demonstrates that molecular dynamics simulation-guided structure-based recombination offers a promising strategy for enhancing the thermostability of other plant enzymes with possible application to increasing the thermotolerance of plants under warming climates., (© 2024 The Author(s). Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Plant glutamate decarboxylase (GAD) is a Ca 2+ -calmodulin (CaM) activated enzyme that produces γ-aminobutyrate (GABA) as the first committed step of the GABA shunt. Our prior research established that in vivo phosphorylation of AtGAD1 (AT5G17330) occurs at multiple N-terminal serine residues following Pi resupply to Pi-starved cell cultures of the model plant Arabidopsis thaliana. The aim of the current investigation was to purify recombinant AtGAD1 (rAtGAD1) following its expression in Escherichia coli to facilitate studies of the impact of phosphorylation on its kinetic properties. However, in vitro proteolytic truncation of an approximate 5 kDa polypeptide from the C-terminus of 59 kDa rAtGAD1 subunits occurred during purification. Immunoblotting demonstrated that most protease inhibitors or cocktails that we tested were ineffective in suppressing this partial rAtGAD1 proteolysis. Although the thiol modifiers N-ethylmaleimide or 2,2-dipyridyl disulfide negated rAtGAD1 proteolysis, they also abolished its GAD activity. This indicates that an essential -SH group is needed for catalysis, and that rAtGAD1 is susceptible to partial degradation either by an E. coli cysteine endopeptidase, or possibly via autoproteolytic activity. The inclusion of exogenous Ca 2+ /CaM facilitated the purification of non-proteolyzed rAtGAD1 to a specific activity of 27 (μmol GABA produced/mg) at optimal pH 5.8, while exhibiting an approximate 3-fold activation by Ca 2+ /CaM at pH 7.3. By contrast, the purified partially proteolyzed rAtGAD1 was >40 % less active at both pH values, and only activated 2-fold by Ca 2+ /CaM at pH 7.3. These results emphasize the need to diagnose and prevent partial proteolysis before conducting kinetic studies of purified regulatory enzymes., Competing Interests: Declaration of Competing interest None declared., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

Pollen development and germination are critical for successful generation of offspring in plants, yet they are highly susceptible to heat stress (HS). However, the molecular mechanism underlying this process has not been fully elucidated. In this study, we highlight the essential roles of two mRNA capping enzymes, named Arabidopsis mRNA capping phosphatase (ARCP) 1 and 2, in regulating male and female gamete development. The transmission efficiencies of gametes carrying arcp1 arcp2 from arcp1 +/- arcp2 -/- and arcp1 -/- arcp2 +/- mutants are 30% and zero, respectively. These mutants exhibited a significant increase in misshaped pollen, with germination rates approximately half of those in wild type. ARCP1/2 exhibit RNA triphosphatase and RNA guanylyltransferase activities, which are required for proper pollen development. Through RNA-seq analysis, genes involved in pollen development/germination and HS response were identified as downregulated genes in pollen from arcp1 +/- arcp2 -/- mutant. Furthermore, ARCP2 protein is degraded under HS condition, and inducing the expression of ARCP2 can increase the pollen germination rate under elevated temperature. We propose that HS triggers the degradation of mRNA capping enzymes, which in turn disrupts the transcriptome that required for pollen development and pollen germination and ultimately leads to male sterility., (© 2024 John Wiley & Sons Ltd.)

4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a crucial herbicide target in current research, playing an important role in the comprehensive management of resistant weeds. However, the limited crop selectivity and less effectiveness against grass weeds of many existing HPPD inhibitors, limit their further application. To address these issues, a series of novel HPPD inhibitors with fused ring structures were designed and synthesized by introducing an electron-rich indazolone ring and combining it with the classical triketone pharmacophore structure. The cocrystal structure of representative compound III-7 complexed with Arabidopsis thaliana HPPD ( At HPPD) was obtained at 2.0 Å resolution to guide the optimization of the designed inhibitor. The optimization results showed that 5-(2-hydroxy-6-oxocyclohex-1-ene-1-carbonyl)-1,4-dimethyl-2-(3-(methylthio)phenyl)-1,2-dihydro-3 H -indazol-3-one, III-15 , was the most active At HPPD inhibitor, with an IC 50 value of 12 nM, nearly 30 times higher efficacy than mesotrione. Greenhouse herbicidal activity tests demonstrated that compound III-15 exhibited excellent herbicidal potency at 30-120 g ai/ha. Notably, it maintained high safety for peanuts even at 120 g ai/ha. Our results showed that compound III-15 is promising as a new candidate HPPD herbicide for use in the peanut fields.

This study was undertaken to develop a high-throughput screening strategy using a whole-cell biosensor to enhance methyl-group transfer, a rate-limiting step influenced by intracellular methyl donor availability and methyltransferase efficiency. An l-homocysteine biosensor was designed based on regulatory protein MetR from Escherichia coli, which rapidly reported intracellular l-homocysteine accumulation resulted from S-adenosyl-l-homocysteine (SAH) formation after methyl-group transfer. Using S-adenosyl-l-methionine (SAM) as a methyl donor, this biosensor was applied to caffeic acid 3-O-methyltransferase derived from Arabidopsis thaliana (AtComT). After several rounds of directed evolution, the modified enzyme achieved a 13.8-fold improvement when converting caffeic acid to ferulic acid. The best mutant exhibited a 5.4-fold improvement in catalytic efficiency. Characterization of beneficial mutants showed that improved O-methyltransferase dimerization greatly contributed to enzyme activity. This finding was verified when we switched and compared the N-termini involved in dimerization across different sources. Finally, with tyrosine as a substrate, the evolved AtComT mutant greatly improved ferulic acid biosynthesis, yielding 3448 mg L -1 with a conversion rate of 88.8%. These results have important implications for high-efficiency O-methyltransferase design, which will greatly benefit the biosynthesis of a wide range of natural products. In addition, the l-homocysteine biosensor has the potential for widespread applications in evaluating the efficiency of SAM-based methyl transfer., 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.)

Chlorogenic acid (CA) is an abundant plant secondary metabolite with promising allelopathic effects on weed growth. However, the molecular targets and mechanism of action of CA in plants remain elusive. Here, we report the employment of a clickable photoaffinity probe in identifying the protein targets of CA in Arabidopsis seedling proteomes. CA specifically binds Arabidopsis dehydroascorbate reductase 1 ( At DHAR1), an enzyme responsible for ascorbate regeneration in plants, by covalent alkylating Cys20 within the catalytic center, thereby inhibiting its activity. In vivo application of CA reduced the pool size and redox state of ascorbate, leading to H 2 O 2 accumulation in Arabidopsis seedlings. In agreement with these results, CA significantly induced the upregulation of antioxidant enzymes and downregulation of proteins involved in water transport and photosynthesis, as evidenced by quantitative proteomics. Taken together, this study revealed DHAR1 as a functional target underlying CA's allelopathic activity in plants, which opens new opportunities for the development of novel herbicides from naturally existing resources.

Cytochrome P450 monooxygenases (P450s) play important roles in herbicide resistance. In this study, there are four amino acid mutations (F39Y, H163Y, S203A, and V361E) between CYP96A146-S and CYP96A146-R , which were cloned, respectively, from susceptible (S) and tribenuron-methyl-resistant (TR) Descurainia sophia . The Arabidopsis expressing CYP96A146-S or CYP96A146-R showed resistance to tribenuron-methyl, carfentrazone-ethyl, and oxyfluorfen, while Arabidopsis transformed with CYP96A146-R or CYP96A146 with any two or three mutations of H163Y, S203A, or V361E exhibited significantly higher resistance to tribenuron-methyl than Arabidopsis expressing CYP96A146-S. The metabolic rates of tribenuron-methyl were significantly faster in Arabidopsis expressing CYP96A146-R than that with CYP96A146-S . The molecular dynamics simulation demonstrated that amino acid mutations did not affect the domain of the HEM ring, which could significantly enhance the volume of the catalytic pocket in P450 (CYP96A146), thereby increasing the collision rate between the catalytic pocket and tribenuron-methyl. Hence, the amino acid mutations may be one of the mechanisms underlying P450-mediated herbicide resistance.

Ethylene is an important plant hormone whose production relies on the action of key enzymes, one of which is 1-aminocyclopropane-1-carboxylate synthase (ACS). There are three classes of ACS, which are all partially regulated by degradation through the ubiquitin-proteasome system (UPS), which regulates ethylene production. Arabidopsis has a single class III ACS, ACS7, but although it is known to be degraded by the 26S proteasome, the UPS proteins involved are poorly characterised. In this work, we used mass spectrometry to identify novel components of the ubiquitin system that may contribute to the regulation of ethylene biosynthesis via ACS7. We found two HECT-type ligases, UPL1 and UPL2, which regulate ACS7 stability. In vitro experiments showed that UPL1 and UPL2 E3 ligases directly control ACS7 turnover. In addition, increased ethylene levels were observed in UPL1- and UPL2-knockout plants in response to NaCl and NaCl+MG132 treatment, respectively. Under the same conditions, we observed increased ACS7 transcript levels in upl1 compared to WT plants under control and stress conditions, further confirming that UPL1 and UPL2 regulate ACS7-dependent ethylene production in response to stress. We used molecular modelling to predict ACS7 ubiquitylation sites and cell-free degradation assays to verify that lysine residues at positions 174, 238 and 384 regulate ACS7 protein stability. Overall, this study provides new insights into the regulation of ACS7 protein stability, and hence ethylene production, in plant growth and development and the response to stress., (© 2025 The Author(s). Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

UDP-glycosyltransferases (UGTs) are the largest glycosyltransferase family developed during the evolution of the plant kingdom. However, their physiological significance in abiotic stress adaptation in land plants is largely unknown. In this study, we identified a UGT gene from Arabidopsis thaliana, UGT86A1, that was significantly induced by salt and drought stresses. To explore the potential biological role of UGT86A1 in salt and drought stress response, we created ugt86a1 knockout mutants and UGT86A1-overexpressing transgenic lines, and analyzed seed germination, seedling development and root growth. The results showed that ugt86a1 mutants are sensitive to drought and salt stresses, while overexpression lines show stronger resistance compared with WT, confirming the positive regulation role of UGT86A1 in abiotic stress response. Our following study indicated that UGT86A1 enhances plant resistance against salt and drought stresses via increasing soluble sugar concentration and promoting ROS scavenging capacity, thereby reducing the damage to plant organs and cells. Moreover, we identified that UGT86A1 largely contributes to the upregulation of multiple stress-induced genes under salt and drought stress conditions. Therefore, our results demonstrated that UGT86A1 is a crucial responsive gene to salt and drought stresses in a land plant, thus promoting the understanding of the physiological significance of the UGT family in plant evolution., (© 2025 Scandinavian Plant Physiology Society.)

Background: Monoacylglycerol lipase (MAGL) belongs to the serine hydrolase family; it catalyzes MAG to produce glycerol and free fatty acids (FFAs), which is the final step in triacylglycerol (TAG) hydrolysis. The effects of MAGL on comprehensive lipid metabolism and plant growth and development have not been elucidated, especially in Arachis hypogaea, an important oil crop., Results: Herein, AhMAGL3b encoding a protein with both hydrolase and acyltransferase regions, a member of MAGL gene family, was cloned and overexpressed in Arabidopsis thaliana. A total of 9 homozygous T 3 generation transgenic lines were obtained. Compared with wild type (WT), overexpression (OE) of AhMAGL3b had no obvious growth inhibition by investigation of agronomic traits, including growth and photosynthetic parameters. The leaf fatty acid (FA) content was increased by 12.1-27.4% in AhMAGL3b-OE lines, while seed oil content was decreased by 10.7-17.3%. Furthermore, the overexpression of AhMAGL3b resulted in higher soluble sugar and starch content, and lower total soluble protein content in both leaves and seeds. Additionally, during seed germination, AhMAGL3b-OE seeds were more dormant than that of WT and the sensitivity to abscisic acid (ABA) treatment was decreased., Conclusions: Taken together, our results indicate that AhMAGL3b is involved in homeostasis among carbohydrates, lipids and protein in A. hypogaea., Competing Interests: Declarations. Ethics approval and consent to participate: All experimental research and field studies on plants were complied with relevant institutional, national, and international guidelines and legislation. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Plant Cysteine Oxidases (PCOs) are oxygen-sensing enyzmes that catalyse oxidation of cysteinyl residues at the N-termini of target proteins, triggering their degradation via the N-degron pathway. PCO oxygen sensitivity means that in low oxygen conditions (hypoxia), their activity reduces and target proteins are stabilised. PCO substrates include Group VII Ethylene Response Factors (ERFVIIs) involved in adaptive responses to the acute hypoxia experienced upon plant submergence, as well as Little Zipper 2 (ZPR2) and Vernalisation 2 (VRN2) which are involved in developmental processes in hypoxic niches. The PCOs are potential targets for improving submergence tolerance through enzyme engineering or chemical treatment. To achieve this, a detailed understanding of their biological function is required. Here, we report development of an assay that exploits the intrinsic fluorescence of Arabidopsis thaliana PCO tryptophan residues. By using Ni(II)-substitued enzymes and preparing the assay under anaerobic conditions, tryptophan fluorescence quenching is observed on enzyme:substrate complex formation, allowing quantification of binding affinities. Our assay revealed that, broadly, AtPCO4 and AtPCO5 have stronger interactions with ERFVII substrates than ZPR2 and VRN2, suggesting ERFVIIs are primary targets of these enzymes. It also revealed a positive cooperative binding effect for interactions between AtPCOs4/5 and ERFVIIs and ZPR2. The assay is experimentally straightforward and can be used to further interogate PCO interactions with substrates., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The vacuole is an important site for RNA degradation. Autophagy delivers RNA to the vacuole, where the vacuolar T2 RNase ribonuclease 2 (RNS2) plays a major role in RNA catabolism. The presumed products of RNS2 activity are 3'-nucleoside monophosphates (3'-NMPs). Vacuolar phosphatases that carry out 3'-NMP hydrolysis are required to metabolize 3'-NMPs, but the specific players remain unknown. Using a mutant of RNS2 and mutants of the autophagy-related genes 5 and 9 (atg5 and atg9), we confirmed that 3'-NMPs are products of vacuolar RNS2-mediated RNA degradation in Arabidopsis (Arabidopsis thaliana). Moreover, we identified purple acid phosphatase 26 (PAP26) and haloacid dehalogenase IIA2.1 (HIIA2.1) as vacuolar 3'-NMP phosphatases. Based on phylogenetic analysis, we propose systematic nomenclature for HADIIA enzymes, some of which were previously named vegetative storage proteins, which we critically discuss. PAP26 and HIIA2.1 differ in their NMP specificity and activity in vitro. However, hiia2.1 pap26 double mutant plants, but generally not the respective single mutants, accumulate 3'-NMPs in addition to 5'-NMPs and, surprisingly, also 2'-NMPs. These findings suggest that PAP26 and HIIA2.1 have overlapping NMP substrate spectra in vivo. Excess 3'- and 2'-NMPs accumulate in plants exposed to a prolonged night, presumably because carbon limitation enhances autophagy-mediated vacuolar RNA degradation. We conclude that vacuolar RNA catabolism releases 3'-NMPs and 2'-NMPs through RNS2 and other RNases that also generate 5'-NMPs. PAP26 and HIIA2.1 are required to dephosphorylate these NMPs, so that they can enter general nucleotide metabolism outside the vacuole., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2025. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Osmotic stress, caused by the lack of water or by high salinity, is a common problem in plant roots. Osmotic stress can be reproducibly simulated with the application of solutions of the high-molecular-weight and impermeable polyethylene glycol. The accumulation of different reactive oxygen species, such as singlet oxygen, superoxide, and hydrogen peroxide, accompany this stress. Among them, singlet oxygen, produced as a byproduct of lipoxygenase activity, has been associated with limiting root growth. To better understand the source and effect of singlet oxygen, we followed its production at the cellular level in Arabidopsis (Arabidopsis thaliana). Osmotic stress initiated profound changes in plastid and vacuole structure. Confocal and electron microscopy showed that the plastids were a source of singlet oxygen accompanied by the appearance of multiple, small extraplastidic bodies that were also an intense source of singlet oxygen. A marker protein, CRUMPLED LEAF, indicated that these small bodies originated from the plastid outer membrane. Remarkably, LINOLEATE 9S-LIPOXYGENASE 5 (LOX5) was shown to change its distribution from uniformly cytoplasmic to a more clumped distribution together with plastids and the small bodies. In addition, oxylipin products of Type 9 lipoxygenase increased, while products of Type 13 lipoxygenases decreased. Inhibition of lipoxygenase by the salicylhydroxamic acid inhibitor or in downregulated lipoxygenase lines prevented cells from initiating the cellular responses, leading to cell death. In contrast, singlet oxygen scavenging halted terminal cell death. These findings underscore the reversible nature of osmotic stress-induced changes, emphasizing the pivotal roles of lipoxygenases and singlet oxygen in root stress physiology., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Salicylic acid (SA) is a prominent defense hormone whose basal level, organ-specific accumulation, and physiological role vary widely among plant species. Of the 2 known pathways of plant SA biosynthesis, the phenylalanine ammonia lyase (PAL) pathway is more ancient and universal but its biosynthetic and physiological roles in diverse plant species remain unclear. Studies in which the PAL pathway is specifically or completely inhibited, as well as a direct comparison of diverse species and different organs within the same species, are needed. To this end, we analyzed the PAL pathway in rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana), 2 distantly related model plants whose basal SA levels and distributions differ tremendously at the organism and tissue levels. Based on our recent identification of the rice peroxisomal cinnamate:CoA ligases (CNLs), we identified 2 peroxisomal CNLs from Arabidopsis and showed CNL as the most functionally specific enzyme among the known enzymes of the PAL pathway. We then revealed the species- and organ-specific contribution of the PAL pathway to benzoic and salicylic acid biosynthesis and clarified its physiological importance in rice and Arabidopsis. Our findings highlight the necessity to consider species and organ types in future SA-related studies and may help to breed new disease-resistant crops., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Key Message: A total of 24 genes of vacuolar H + -translocating pyrophosphatases H + -PPases (VPP) genes were identified in Saccharum spontaneum AP85-441 and the ScVPP1-overexpressed Arabidopsis plants conferred salt tolerance. The vital role of vacuolar H + -translocating pyrophosphatases H + -PPases (VPP) genes involved in plants in response to abiotic stresses. However, the understanding of VPP functions in sugarcane remained unclear. In this study, a total of 24 VPP genes (SsaVPP1-SsaVPP24) were identified in the Saccharum spontaneum genome of haploid clone AP85-441. These genes were distributed in two phylogenetic groups. The SsaVPPs displayed diverse physio-chemical and gene structure attributes. The SsaVPP family genes have expanded through segmental duplication (20 gene pairs) rather than tandem duplication. A full-length cDNA of ScVPP1 was cloned from the sugarcane cultivar ROC22 and shared 99.48% sequence identity (amino acid) with homologous gene SsaVPP21 from AP85-441. In ROC22, the ScVPP1 gene was considerably upregulated by NaCl and ABA treatments among leaf, root, and stem tissues, while this gene was exclusively upregulated in the root with PEG treatment. Under NaCl and ABA stresses, yeast cells transfected by the ScVPP1 plasmid showed distinct growth rates compared to control yeast cells transfected by the empty vector. In transgenic Arabidopsis lines overexpressing ScVPP1, the seed gemination and survival rate were enhanced under NaCl treatment but not under ABA stress as compared to wild-type plants. These results suggested that the ScVPP1 gene conferred tolerance to slat and may be used as a salt resistance gene source for sugarcane breeding., Competing Interests: Declarations. Conflict of interest: The authors declare no conflicts of interest., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Lysophosphatidic acid acyltransferase1 (LPAT1) catalyzes the second step of de novo glycerolipid biosynthesis in chloroplasts. However, the embryonic-lethal phenotype of the knockout mutant suggested an unknown role for LPAT1 in non-photosynthetic reproductive organs. Reciprocal genetic crossing of the lpat1-1 heterozygous line suggested a female gametophytic defect of the lpat1-1 knockout mutant. By suppressing LPAT1 specifically during seed development, we showed that LPAT1 suppression affected silique growth and seed production. Glycerolipid analysis of the LPAT1 knockdown lines revealed a pronounced decrease of phosphatidylcholine (PC) content in mature siliques along with an altered polyunsaturation level of the polar glycerolipids. In seeds, the acyl composition of triacylglycerol (TAG) was altered albeit not the content. These results indicate that plastidic LPAT1 plays an important role in reproductive growth and extraplastidic glycerolipid metabolism involving PC and TAG., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Oriented cell divisions establish plant tissue and organ patterning and produce different cell types; this is particularly true of the highly organized Arabidopsis (Arabidopsis thaliana) root meristem. Mutant alleles of INFLORESCENCE AND ROOT APICES RECEPTOR KINASE (IRK) exhibit excess cell divisions in the root endodermis. IRK is a transmembrane receptor kinase that localizes to the outer polar domain of these cells, suggesting that directional signal perception is necessary to repress endodermal cell division. Here, a detailed examination revealed many of the excess endodermal divisions in irk have division planes that specifically skew toward the outer lateral side. Therefore, we termed them "outward askew" divisions. Expression of an IRK truncation lacking the kinase domain retains polar localization and prevents outward askew divisions in irk; however, the roots exhibit excess periclinal endodermal divisions. Using cell identity markers, we show that the daughters of outward askew divisions transition from endodermal to cortical identity similar to those of periclinal divisions. These results extend the requirement for IRK beyond repression of cell division activity to include cell division plane positioning. Based on its polarity, we propose that IRK at the outer lateral endodermal cell face participates in division plane positioning to ensure normal root ground tissue patterning., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Arginine is an important amino acid in plants, as it not only plays a structural role and serves as nitrogen storage but is also a precursor for various molecules, including polyamines and proline. Arginine is produced by argininosuccinate lyase (ASL) which catalyzes the cleavage of argininosuccinate to arginine and fumarate. ASL belongs to the fumarate lyase family and while many members of this family were well-characterized, little is known about plant ASLs. Here we present the first crystal structures of ASL from the model plant, Arabidopsis thaliana (AtASL). One of the structures represents the unliganded form of the AtASL homotetramer. The other structure, obtained from a crystal soaked in argininosuccinate, accommodates the substrate or the reaction products in one of four active sites of the AtASL tetramer. Each active site is located at the interface of three neighboring protomers. The AtASL structure with ligands allowed us to analyze the enzyme-substrate and the enzyme-product interactions in detail. Furthermore, based on our analyses, we describe residues of AtASL crucial for catalysis. The structure of AtASL gives the rationale for the open-to-close transition of the GSS mobile loop and indicates the importance of serine 333 from this loop for the enzymatic action of the enzyme. Finally, we supplemented the structural data with the identification of sequence motifs characteristic for ASLs., 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 The Author(s). Published by Elsevier Inc. All rights reserved.)

Phosphate (Pi) homeostasis is important for plant growth and adaptation to the dynamic environment, which requires the precise regulation of phosphate transporter (PHT) trafficking from the endoplasmic reticulum to the plasma membrane. LIPOYL SYNTHASE 1p (LIP1p) is known as a key enzyme in plastids to catalyze lipoylation of pyruvate dehydrogenase complex for de novo fatty acid synthesis. It is unknown whether this process is involved in regulating Pi homeostasis. Here, we demonstrate a new role of LIP1p in controlling Pi homeostasis by regulating PHT1 trafficking. We recovered a weak mutant allele of LIP1p in Arabidopsis that accumulates much less Pi and has enhanced expression of phosphate starvation-induced genes. LIP1p mutation alters the lipid profile and compromises vesicle trafficking of PHT1 to the plasma membrane to impair Pi uptake. Beside phosphorus, the homeostasis of a series of mineral nutrients was also perturbed in lip1p mutant. Our findings provide powerful genetic evidence to support the linkage between lipoylation and ion homeostasis in plants., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Plant receptor kinases (RKs) are critical for transmembrane signalling involved in various biological processes including plant immunity. MALE DISCOVERER1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2) is a unique RK that recognizes a family of immunomodulatory peptides called SERINE-RICH ENDOGENOUS PEPTIDEs (SCOOPs) and activates pattern-triggered immunity responses. However, the precise mechanisms underlying SCOOP recognition and activation of MIK2 remain poorly understood. Here we present the cryogenic electron microscopy structure of a ternary complex consisting of the extracellular leucine-rich repeat (LRR) of MIK2 (MIK2LRR), SCOOP12 and the extracellular LRR of the co-receptor BAK1 (BAK1LRR) at a resolution of 3.34 Å. The structure reveals that a DNHH motif in MIK2LRR plays a critical role in specifically recognizing the highly conserved SxS motif of SCOOP12. Furthermore, the structure demonstrates that N-glycans at MIK2LRR Asn410 directly interact with the N-terminal capping region of BAK1LRR. Mutation of the glycosylation site, MIK2LRR N410D , completely abolishes the SCOOP12-independent interaction between MIK2LRR and BAK1LRR and substantially impairs the assembly of the MIK2LRR-SCOOP12-BAK1LRR complex. Supporting the biological relevance of N410-glycosylation, MIK2 N410D substantially compromises SCOOP12-triggered immune responses in plants. Collectively, these findings elucidate the mechanism underlying the loose specificity of SCOOP recognition by MIK2 and reveal an unprecedented mechanism by which N-glycosylation modification of LRR-RK promotes receptor activation., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Activity-based sensing probes are powerful tools for monitoring enzymatic activities in complex biological samples such as cellular and live animals; however, their application in plants remains challenging. Herein, fourteen activity-based fluorescent probes were assayed against Arabidopsis O-methyltransferases (AtOMTs). One probe, 3-BTD, displayed a high selectivity, reactivity, and fluorescence response toward AtOMTs especially the isoform AtCCoAOMT. We further characterized the features of this probe and explored whether it could be used to detect OMT activities in living plant cells. Our results show that 3-BTD can be used to visualize OMT activity in Arabidopsis, and no fluorescent signal was observed in the comt/ccoaomt double mutant, indicating that it has good specificity. Interestingly, in contrast to the observation that AtCCoAOMT-YFP accumulated in both cytoplasm and nucleus, OMT enzymatic activity tracked by 3-BTD probe was found only in the cytoplasm. This underscores the importance of activity-based sensing in studying protein function. Moreover, 3-BTD can be successfully applied in OMT visualization of different plants. This study indicates that 3-BTD can serve as a potential probe for in situ monitoring the real activity of OMT in multiple plants and provides a strategy for visualizing the activity of other enzymes in plants., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Fatty acid desaturase (FAD) is essential for plant growth and development and plant defence response. Although flax (Linum usitatissimum L.) is an important oil and fibre crop, but its FAD gene remains understudied. This study identified 43 LuFAD genes in the flax genome. The phylogenetic analysis divided the FAD genes into seven subfamilies. LuFAD is unevenly distributed on 15 chromosomes, and fragment duplication is the only driving force for the amplification of the LuFAD gene family. In the LuFAD gene promoter region, most elements respond to plant hormones (MeJA, ABA) and abiotic stresses (anaerobic and low temperature). The expression pattern analysis showed that the temporal and spatial expression patterns of all LuFAD genes in different tissues and the response patterns to abiotic stresses (heat and salt) were identified. Subcellular localisation showed that all LuFAD2-GFP were expressed in the endoplasmic reticulum membrane. RT-qPCR analysis revealed that LuFAD2 was significantly upregulated under cold, salt and drought stress, and its overexpression in Arabidopsis thaliana enhanced cold tolerance genes and reduced ROS accumulation. This study offers key insights into the FAD gene family's role in flax development and stress adaptation., (© 2024 The Author(s). Plant, Cell & Environment published by John Wiley & Sons Ltd.)

The committed step for de novo fatty acid (FA) synthesis is the ATP-dependent carboxylation of acetyl-coenzyme A catalysed by acetyl-CoA carboxylase (ACCase). In most plants, ACCase is a multi-subunit complex orthologous to prokaryotes. However, unlike prokaryotes, the plant and algal orthologues are comprised both catalytic and additional dedicated regulatory subunits. Novel regulatory subunits, biotin lipoyl attachment domain-containing proteins (BADC) and carboxyltransferase interactors (CTI) (both three-gene families in Arabidopsis ) represent new effectors specific to plants and certain algal species. The evolutionary history of these genes in autotrophic eukaryotes remains elusive, making it an ongoing area of research. Analyses of potential protein-protein and co-occurrence interactions, informed by gene network patterns using the STRING database, in Arabidopsis thaliana and Chlamydomonas reinhardtii unveil intricate gene associations with ACCase, suggesting a complex interplay between FA synthesis and other cellular processes. Among both species, a higher number of co-expressed genes was identified in Arabidopsis , indicating a wider potential regulatory network of ACCase in plants. This review investigates the extent to which these genes arose in autotrophic eukaryotes and provides insights into their evolutionary trajectory. This article is part of the theme issue 'The evolution of plant metabolism'.

Auxins play a critical role in several plant developmental processes and their endogenous levels are regulated at multiple levels. The enzymes of the GRETCHEN HAGEN 3 (GH3) protein family catalyze the conjugation of amino acids to indoleacetic acid (IAA), the major endogenous auxin. The GH3 proteins are encoded by multiple redundant genes in plant genomes, making it difficult to perform functional genetic studies to understand their role in auxin homeostasis. To address these challenges, we used a chemical approach that exploits the reaction mechanism of GH3 proteins to identify small molecule inhibitors of their activity from a defined chemical library. The study evaluated receptor-ligand complexes based on their binding energy and classified them accordingly. Docking algorithms were used to correct any deviations, resulting in a list of the most important inhibitory compounds for selected GH3 enzymes based on a normalized sum of energy. The study presents atomic details of protein-ligand interactions and quantifies the effect of several of the identified small molecule inhibitors on auxin-mediated root growth processes in Arabidopsis thaliana. The direct effect of these compounds on endogenous auxin levels was measured using appropriate auxin sensors and endogenous hormone measurements. Our study has identified novel compounds of the flavonoid biosynthetic pathway that are effective inhibitors of GH3 enzyme-mediated IAA conjugation. These compounds play a versatile role in hormone-regulated plant development and have potential applications in both basic research and agriculture., (© 2024 Scandinavian Plant Physiology Society.)

Polysaccharide structural complexity not only influences cell wall strength and extensibility but also hinders pathogenic and biotechnological attempts to saccharify the wall. In certain species and tissues, glucuronic acid side groups on xylan exhibit arabinopyranose or galactose decorations whose genetic and evolutionary basis is completely unknown, impeding efforts to understand their function and engineer wall digestibility. Genetics and polysaccharide profiling were used to identify the responsible loci in Arabidopsis and Eucalyptus from proposed candidates, while phylogenies uncovered a shared evolutionary origin. GH30-family endo-glucuronoxylanase activities were analysed by electrophoresis, and their differing specificities were rationalised by phylogeny and structural analysis. The newly identified xylan arabinopyranosyltransferases comprise an overlooked subfamily in the GT47-A family of Golgi glycosyltransferases, previously assumed to comprise mainly xyloglucan galactosyltransferases, highlighting an unanticipated adaptation of both donor and acceptor specificities. Further neofunctionalisation has produced a Myrtaceae-specific xylan galactosyltransferase. Simultaneously, GH30 endo-glucuronoxylanases have convergently adapted to overcome these decorations, suggesting a role for these structures in defence. The differential expression of glucuronoxylan-modifying genes across Eucalyptus tissues, however, hints at further functions. Our results demonstrate the rapid adaptability of biosynthetic and degradative carbohydrate-active enzyme activities, providing insight into plant-pathogen interactions and facilitating plant cell wall biotechnological utilisation., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Salt stress severely affects the growth and yield of apples (Malus domestica Borkh). Although salt-tolerant genes have been extensively studied, documentation on the role of the ATP-dependent phosphofructokinase gene MdPFK5 in salt stress is limited. This study conducted an evolutionary tree and three-dimensional structure analysis of the PFK gene family in Arabidopsis thaliana and MdPFK (MD01G1037400), revealing a close phylogenetic relationship between MdPFK (MD01G1037400) and AtPFK5. Given the similarity in their protein tertiary structures, MdPFK was designated as MdPFK5, suggesting functional similarities with AtPFK5. Further investigation revealed elevated expression levels of MdPFK5 in apple leaves and flowers, particularly showing significant upregulation 120 days after blooming and differential expression beginning at 3 hours of salt stress. Overexpression of MdPFPK5 conferred salt tolerance in both apple calli and transgenic lines of Arabidopsis thaliana. Moreover, NaCl treatment promoted soluble sugar accumulation in apple calli and transgenic lines of Arabidopsis thaliana overexpressing MdPFK5. This study provides new insights into the salt tolerance function of MdPFK5., (© 2024 Scandinavian Plant Physiology Society.)

In angiosperms, ovules give rise to seeds upon fertilization. Thus, seed formation is dependent on both successful ovule development and tightly controlled communication between female and male gametophytes. During establishment of these interactions, cell walls play a pivotal role, especially arabinogalactan-proteins (AGPs). AGPs are highly glycosylated proteins decorated by arabinogalactan side chains, representing 90 % of the AGP molecule. AGP glycosylation is initiated by a reaction catalysed by hydroxyproline-O-galactosyltransferases (Hyp-GALTs), specifically eight of them (GALT2-9), which add the first galactose to Hyp residues. Five Hyp-GALTs (GALT2, 5, 7, 8 and 9) were previously described as essential for AGP functions in pollen and ovule development, pollen-pistil interactions, and seed morphology. In the present work, a higher order Hyp-GALT mutant (23456789) was studied, with a high degree of under-glycosylated AGPs, to gain deeper insight into the crucial roles of these eight enzymes in female reproductive tissues. Notably, the 23456789 mutant demonstrated a high quantity of unfertilized ovules, displaying abnormal callose accumulation both at the micropylar region and, sometimes, throughout the entire embryo sac. Additionally, this mutant displayed ovules with abnormal embryo sacs, had a disrupted spatiotemporal distribution of AGPs in female reproductive tissues, and showed abnormal seed and embryo development, concomitant with a reduction in AGP-GlcA levels. This study revealed that at least three more enzymes exhibit Hyp-O-GALT activity in Arabidopsis (GALT3, 4 and 6), and reinforces the crucial importance of AGP carbohydrates in carrying out the biological functions of AGPs during plant reproduction., 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 The Authors. Published by Elsevier B.V. All rights reserved.)

Arabidopsis uracil phosphoribosyltransferase (UPP) is an essential enzyme and plants lacking this enzyme are strongly compromised in chloroplast function. Our analysis of UPP amiRNA mutants has confirmed that this vital function is crucial to establish a fully functional photosynthesis as the RIESKE iron sulfur protein (PetC) is almost absent, leading to a block in photosynthetic electron transport. Interestingly, this function appears to be unrelated to nucleotide homeostasis since nucleotide levels were not altered in the studied mutants. Transcriptomics and proteomic analysis showed that protein homeostasis but not gene expression is most likely responsible for this observation and high light provoked an upregulation of protease levels, including thylakoid filamentation temperature-sensitive 1, 5 (FtsH), caseinolytic protease proteolytic subunit 1 (ClpP1), and processing peptidases, as well as components of the chloroplast protein import machinery in UPP amiRNA lines. Strongly reduced PetC amounts were not only detected by immunoblot from mature plants but in addition in a de-etiolation experiment with young seedlings and are causing reduced high light-induced non-photochemical quenching Φ (NPQ) but increased unregulated energy dissipation Φ (NO) . This impaired photosynthesis results in an inability to induce flavonoid biosynthesis. In addition, the levels of the osmoprotectants raffinose, proline, and fumarate were found to be reduced. In sum, our work suggests that UPP assists in stabilization PetC during import, processing or targeting to the thylakoid membrane, or protects it against proteolytic degradation., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

The oxidative pentose phosphate (OPP) pathway provides metabolic intermediates for the shikimate pathway and directs carbon flow to the biosynthesis of aromatic amino acids (AAAs), which serve as basic protein building blocks and precursors of numerous metabolites essential for plant growth. However, genetic evidence linking the two pathways is largely unclear. In this study, we identified 6-phosphogluconate dehydrogenase 2 (PGD2), the rate-limiting enzyme of the cytosolic OPP pathway, through suppressor screening of arogenate dehydrogenase 2 (adh2) in Arabidopsis. Our data indicated that a single amino acid substitution at position 63 (glutamic acid to lysine) of PGD2 enhanced its enzyme activity by facilitating the dissociation of products from the active site of PGD2, thus increasing the accumulation of AAAs and partially restoring the defective phenotype of adh2. Phylogenetic analysis indicated that the point mutation occurred in a well-conserved amino acid residue. Plants with different amino acids at this conserved site of PGDs confer diverse catalytic activities, thus exhibiting distinct AAAs producing capability. These findings uncover the genetic link between the OPP pathway and AAAs biosynthesis through PGD2. The gain-of-function point mutation of PGD2 identified here could be considered as a potential engineering target to alter the metabolic flux for the production of AAAs and downstream compounds., (© 2024. Science China Press.)

Lignin is a phenolic polymer that is a major source of biomass. Oxidative enzymes, such as laccase and peroxidase, are required for lignin polymerisation. Laccase is a member of the multicopper oxidase family and has a high amino acid sequence similarity with ascorbate oxidase. However, the process of functional differentiation between the two enzymes remains poorly understood. In this study, the common ancestry sequence of laccase and ascorbate oxidase (AncMCO) was predicted via phylogenetic reconstruction, and its in vivo effect on lignin biosynthesis in Arabidopsis thaliana was assessed. The estimated AncMCO sequence conserved key residues that coordinate with copper ions, implying that the electron transfer system is likely to be conserved in AncMCO. However, multiple insertions/deletions corresponding to protein surface structures have been found between laccase, ascorbate oxidase, and AncMCO. The overexpression of canonical laccase (AtLAC4) and ascorbate oxidase (AtAAO1) in A. thaliana resulted in notable increases of syringyl/guaiacyl lignin unit ratio in stems, whereas, in contrast, the overexpression of AncMCO did not show any detectable change in lignin deposition. Transcriptomic analysis revealed that the AtAAO1-overexpressing line exhibited significant changes in the expression of a wide range of cell wall biosynthesis genes. These results highlight the importance of the molecular evolution of multicopper oxidase, which drives lignin biosynthesis during plant evolution., (© 2024. The Author(s) under exclusive licence to The Botanical Society of Japan.)

Carbonic anhydrases (CAs) are the main enzymes handling bicarbonate in the different cell compartments. This study analyses the expression of CAs in roots of Arabidopsis thaliana demes differing in tolerance to bicarbonate: the tolerant A1 (C+) deme and the sensitive deme, T6 (C-) . Exposure to 10 mM NaCl caused a transient depolarization of the root cell membranes, and in contrast, the supply of 10 mM NaHCO 3 caused hyperpolarization. This hyperpolarization was much stronger in A1 (C+) than in T6 (C-) . Acetazolamide (AZ), a specific inhibitor of CAs, abolished the hyperpolarizing effect in A1 (C+) , indicating the implication of CAs in this fast membrane response. The time-dependent (3 to 72 h) expression profiles of 14 CAs in roots of A1 (C+) and T6 (C-) exposed to either control (0 mM NaHCO 3 , pH 5.9), or bicarbonate (10 mM NaHCO 3 ,pH 8.3) conditions revealed a bicarbonate specific upregulation of BCA4.1 (from 3 to 12 h) in A1 (C+) . Contrastingly, in T6 (C-) BCA4.1 was downregulated by NaHCO 3 . Exclusively in A1 (C+) , the enhanced expression of BCA4.1 under bicarbonate was parallelled by an increase of PIP1,3, SLAH1, SLAH3, AHA2, and FRO2 gene expression levels. Under HCO 3 - exposure, a bca4 knockout mutant had a lower number of lateral roots, lower root diameters, and higher root lipid peroxidation than the WT. These results indicate that bicarbonate-induced root membrane hyperpolarization is the fast (minutes) initial signalling event in the tolerance response. This is followed by the specific upregulation of BCA4.1 and genes involved in H 2 O and CO 2 transport, apoplast acidification, and iron acquisition., (© 2024 The Author(s). Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.)

Key Message: The N-terminal transmembrane domain of LPAT1 crosses the inner membrane placing the N terminus in the intermembrane space and the C-terminal enzymatic domain in the stroma. Galactolipids mono- and di-galactosyl diacylglycerol are the major and vital lipids of photosynthetic membranes. They are synthesized by five enzymes hosted at different sub-chloroplast locations. However, localization and topology of the second-acting enzyme, lysophosphatidic acid acyltransferase 1 (LPAT1), which acylates the sn-2 position of glycerol-3-phosphate (G3P) to produce phosphatidic acid (PA), remain unclear. It is not known whether LPAT1 is located at the outer or the inner envelope membrane and whether its enzymatic domain faces the cytosol, the intermembrane space, or the stroma. Even the size of mature LPAT1 in chloroplasts is not known. More information is essential for understanding the pathways of metabolite flow and for future engineering endeavors to modify glycerolipid biosynthesis. We used LPAT1 preproteins translated in vitro for import assays to determine the precise size of the mature protein and found that the LPAT1 transit peptide is at least 85 residues in length, substantially longer than previously predicted. A construct comprising LPAT1 fused to the Venus fluorescent protein and driven by the LPAT1 promoter was used to complement an Arabidopsis lpat1 knockout mutant. To determine the sub-chloroplast location and topology of LPAT1, we performed protease treatment and alkaline extraction using chloroplasts containing in vitro-imported LPAT1 and chloroplasts isolated from LPAT1-Venus-complemented transgenic plants. We show that LPAT1 traverses the inner membrane via an N-terminal transmembrane domain, with its N terminus protruding into the intermembrane space and the C-terminal enzymatic domain residing in the stroma, hence displaying a different membrane topology from its bacterial homolog, PlsC., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Salicylic acid (SA) plays a crucial role in plant defense against biotrophic and semibiotrophic pathogens. In Arabidopsis (Arabidopsis thaliana), isochorismate synthase 1 (AtICS1) is a key enzyme for the pathogen-induced biosynthesis of SA via catalytic conversion of chorismate into isochorismate, an essential precursor for SA synthesis. Despite the extensive knowledge of ICS1-related menaquinone, siderophore, and tryptophan (MST) enzymes in bacteria, the structural mechanisms for substrate binding and catalysis in plant isochorismate synthase (ICS) enzymes are unknown. This study reveals that plant ICS enzymes catalyze the isomerization of chorismate through a magnesium-dependent mechanism, with AtICS1 exhibiting the most substantial catalytic activity. Additionally, we present high-resolution crystal structures of apo AtICS1 and its complex with chorismate, offering detailed insights into the mechanisms of substrate recognition and catalysis. Importantly, our investigation indicates the existence of a potential substrate entrance channel and a gating mechanism regulating substrate into the catalytic site. Structural comparisons of AtICS1 with MST enzymes suggest a shared structural framework with conserved gating and catalytic mechanisms. This work provides valuable insights into the structural and regulatory mechanisms governing substrate delivery and catalysis in AtICS1, as well as other plant ICS enzymes., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Xylan is one of the major hemicelluloses in plant cell walls and its xylosyl backbone is often decorated at O-2 with glucuronic acid (GlcA) and/or methylglucuronic acid (MeGlcA) residues. The GlcA/MeGlcA side chains may be further substituted with 2-O-arabinopyranose (Arap) or 2-O-galactopyranose (Gal) residues in some plant species, but the enzymes responsible for these substitutions remain unknown. During our endeavor to investigate the enzymatic activities of Arabidopsis MUR3-clade members of the GT47 glycosyltransferase family, we found that one of them was able to transfer Arap from UDP-Arap onto O-2 of GlcA side chains of xylan, and thus it was named xylan 2-O-arabinopyranosyltransferase 1 (AtXAPT1). The function of AtXAPT1 was verified in planta by its T-DNA knockout mutation showing a loss of the Arap substitution on xylan GlcA side chains. Further biochemical characterization of XAPT close homologs from other plant species demonstrated that while the poplar ones had the same catalytic activity as AtXAPT1, those from Eucalyptus, lemon-scented gum, sea apple, 'Ohi'a lehua, duckweed and purple yam were capable of catalyzing both 2-O-Arap and 2-O-Gal substitutions of xylan GlcA side chains albeit with differential activities. Sequential reactions with XAPTs and glucuronoxylan methyltransferase 3 (GXM3) showed that XAPTs acted poorly on MeGlcA side chains, whereas GXM3 could efficiently methylate arabinosylated or galactosylated GlcA side chains of xylan. Furthermore, molecular docking and site-directed mutagenesis analyses of Eucalyptus XAPT1 revealed critical roles of several amino acid residues at the putative active site in its activity. Together, these findings establish that XAPTs residing in the MUR3 clade of family GT47 are responsible for 2-O-arabinopyranosylation and 2-O-galactosylation of GlcA side chains of xylan., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Plastid-encoded RNA polymerase (PEP) is a bacterial-type multisubunit RNA polymerase responsible for the majority of transcription in chloroplasts. PEP consists of four core subunits, which are orthologs of their cyanobacterial counterparts. In Arabidopsis thaliana, PEP is expected to interact with 14 PEP-associated proteins (PAPs), which serve as peripheral subunits of the RNA polymerase. The exact contributions of PAPs to PEP function are still poorly understood. We used ptChIP-seq to show that PAP1 (also known as pTAC3), a peripheral subunit of PEP, binds to the same genomic loci as RpoB, a core subunit of PEP. The pap1 mutant shows a complete loss of RpoB binding to DNA throughout the genome, indicating that PAP1 is necessary for RpoB binding to DNA. A similar loss of RpoB binding to DNA is observed in a mutant defective in PAP7 (also known as pTAC14), another peripheral PEP subunit. We propose that PAPs are required for the recruitment of core PEP subunits to DNA., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Ethylamine (EA), the precursor of theanine biosynthesis, is synthesized from alanine decarboxylation by alanine decarboxylase (AlaDC) in tea plants. AlaDC evolves from serine decarboxylase (SerDC) through neofunctionalization and has lower catalytic activity. However, lacking structure information hinders the understanding of the evolution of substrate specificity and catalytic activity. In this study, we solved the X-ray crystal structures of AlaDC from Camellia sinensis (CsAlaDC) and SerDC from Arabidopsis thaliana (AtSerDC). Tyr 341 of AtSerDC or the corresponding Tyr 336 of CsAlaDC is essential for their enzymatic activity. Tyr 111 of AtSerDC and the corresponding Phe 106 of CsAlaDC determine their substrate specificity. Both CsAlaDC and AtSerDC have a distinctive zinc finger and have not been identified in any other Group II PLP-dependent amino acid decarboxylases. Based on the structural comparisons, we conducted a mutation screen of CsAlaDC. The results indicated that the mutation of L110F or P114A in the CsAlaDC dimerization interface significantly improved the catalytic activity by 110% and 59%, respectively. Combining a double mutant of CsAlaDC L110F/P114A with theanine synthetase increased theanine production 672% in an in vitro system. This study provides the structural basis for the substrate selectivity and catalytic activity of CsAlaDC and AtSerDC and provides a route to more efficient biosynthesis of theanine., Competing Interests: HW, BZ, SQ, CD, XW, WG, ZZ No competing interests declared, (© 2023, Wang, Zhu et al.)

Main Conclusion: This review focuses on HATs and HDACs that modify non-histone proteins, summarizes functional mechanisms of non-histone acetylation as well as the roles of HATs and HDACs in rice and Arabidopsis. The growth and development of plants, as well as their responses to biotic and abiotic stresses, are governed by intricate gene and protein regulatory networks, in which epigenetic modifying enzymes play a crucial role. Histone lysine acetylation levels, modulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), are well-studied in the realm of transcriptional regulation. However, the advent of advanced proteomics has unveiled that non-histone proteins also undergo acetylation, with its underlying mechanisms now being clarified. Indeed, non-histone acetylation influences protein functionality through diverse pathways, such as modulating protein stability, adjusting enzymatic activity, steering subcellular localization, influencing interactions with other post-translational modifications, and managing protein-protein and protein-DNA interactions. This review delves into the recent insights into the functional mechanisms of non-histone acetylation in plants. We also provide a summary of the roles of HATs and HDACs in rice and Arabidopsis, and explore their potential involvement in the regulation of non-histone proteins., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The action of abscisic acid (ABA) is closely related to its level in plant tissues. Uridine diphosphate-glycosyltransferase71c5 (UGT71C5) was characterized as a major UGT enzyme to catalyze the formation of the ABA-glucose ester (ABA-GE), a reversible inactive form of free ABA in Arabidopsis thaliana (thale cress). UGTs function in a mode where the catalytic base deprotonates an acceptor to allow a nucleophilic attack at the anomeric center of the donor, achieving the transfer of a glucose moiety. The proteomic data revealed that UGT71C5 can be persulfidated. Herein, an experimental method was employed to detect the persulfidation site of UGT71C5, and the computational methods were further used to identify the yet unknown molecular basis of ABA glycosylation as well as the regulatory role of persulfidation in this process. Our results suggest that the linker and the U-shaped loop are regulatory structural elements: the linker is associated with the binding of uridine diphosphate glucose (UPG) and the U-shaped loop is involved in binding both UPG and ABA.It was also found that it is through tuning the dynamics of the U-shaped loop that is accompanied by the movement of tyrosine (Y388) that the persulfidation of cysteine (C311) leads to the catalytic residue histidine (H16) being in place, preparing for the deprotonation of ABA, and then reorientates UPG and deprotonated ABA closer to the 'Michaelis' complex, facilitating the transfer of a glucose moiety. Ultimately, the persulfidation of UGT71C5 is in favor of ABA glycosylation. Our results provide insights into the molecular details of UGT71C5 recognizing substrates and insights concerning persulfidation as a possible mechanism for hydrogen sulfide (H 2 S) to modulate the content of ABA, which helps us understand how modulating ABA level strengthens plant tolerance.

Stress granules (SGs) are conserved reversible cytoplasmic condensates enriched with aggregation-prone proteins assembled in response to various stresses. How plants regulate SG dynamics is unclear. Here, we show that 26S proteasome is a stable component of SGs, promoting the overall clearance of SGs without affecting the molecular mobility of SG components. Increase in either temperature or duration of heat stress reduces the molecular mobility of SG marker proteins and suppresses SG clearance. Heat stress induces dramatic ubiquitylation of SG components and enhances the activities of SG-resident proteasomes, allowing the degradation of SG components even during the assembly phase. Their proteolytic activities enable the timely disassembly of SGs and secure the survival of plant cells during the recovery from heat stress. Therefore, our findings identify the cellular process that de-couples macroscopic dynamics of SGs from the molecular dynamics of its constituents and highlights the significance of the proteasomes in SG disassembly., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The metabolism of tetrahydrofolate (H 4 PteGlu n )-bound one-carbon (C 1 ) units (C 1 metabolism) is multifaceted and required for plant growth, but it is unclear what of many possible synthesis pathways provide C 1 units in specific organelles and tissues. One possible source of C 1 units is via formate-tetrahydrofolate ligase, which catalyzes the reversible ATP-driven production of 10-formyltetrahydrofolate (10-formyl-H 4 PteGlu n ) from formate and tetrahydrofolate (H 4 PteGlu n ). Here, we report biochemical and functional characterization of the enzyme from Arabidopsis thaliana (AtFTHFL). We show that the recombinant AtFTHFL has lower K m and k cat values with pentaglutamyl tetrahydrofolate (H 4 PteGlu 5 ) as compared to monoglutamyl tetrahydrofolate (H 4 PteGlu 1 ), resulting in virtually identical catalytic efficiencies for the two substrates. Stable transformation of Arabidopsis plants with the EGFP-tagged AtFTHFL, followed with fluorescence microscopy, demonstrated cytosolic signal. Two independent T-DNA insertion lines with impaired AtFTHFL function had shorter roots compared to the wild type plants, demonstrating the importance of this enzyme for root growth. Overexpressing AtFTHFL led to the accumulation of H 4 PteGlu n  + 5,10-methylene-H 4 PteGlu n and serine, accompanied with the depletion of formate and glycolate, in roots of the transgenic Arabidopsis plants. This metabolic adjustment supports the hypothesis that AtFTHFL feeds the cytosolic C 1 network in roots with C 1 units originating from glycolate, and that these units are then used mainly for biosynthesis of serine, and not as much for the biosynthesis of 5-methyl-H 4 PteGlu n , methionine, and S-adenosylmethionine. This finding has implications for any future attempts to engineer one-carbon unit-requiring products through manipulation of the one-carbon metabolic network in non-photosynthetic organs., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Mitochondrial respiration is major source of chemical energy for all free-living eukaryotes. Nevertheless, the mechanisms of the respiratory complexes and supercomplexes remain poorly understood. Here, I review recent structural and functional investigations of plant supercomplex I + III2 from Arabidopsis thaliana and Vigna radiata. I discuss commonalities, open questions and implications for complex I, complex III2 and supercomplexes in plants and non-plants. Studies across further clades will enhance our understanding of respiration and the potential universal mechanisms of its complexes and supercomplexes., (© 2024 The Author(s).)

Meiosis is a critical process in sexual reproduction, and errors during this cell division can significantly impact fertility. Successful meiosis relies on the coordinated action of numerous genes involved in DNA replication, strand breaks, and subsequent rejoining. DNA topoisomerase enzymes play a vital role by regulating DNA topology, alleviating tension during replication and transcription. To elucidate the specific function of DNA topoisomerase 1α ( A t T O P 1 α ) in male reproductive development of Arabidopsis thaliana , we investigated meiotic cell division in Arabidopsis flower buds. Combining cytological and biochemical techniques, we aimed to reveal the novel contribution of A t T O P 1 α to meiosis. Our results demonstrate that the absence of A t T O P 1 α leads to aberrant chromatin behavior during meiotic division. Specifically, the top1α1 mutant displayed altered heterochromatin distribution and clustered centromere signals at early meiotic stages. Additionally, this mutant exhibited disruptions in the distribution of 45s rDNA signals and a reduced frequency of chiasma formation during metaphase I, a crucial stage for genetic exchange. Furthermore, the atm-2×top1α1 double mutant displayed even more severe meiotic defects, including incomplete synapsis, DNA fragmentation, and the presence of polyads. These observations collectively suggest that A t T O P 1 α plays a critical role in ensuring accurate meiotic progression, promoting homologous chromosome crossover formation, and potentially functioning in a shared DNA repair pathway with ATAXIA TELANGIECTASIA MUTATED (ATM) in Arabidopsis microspore mother cells., Competing Interests: Diaa Abd El-Moneim is an Academic Editor for PeerJ., (© 2024 Elesawi et al.)

Metabolic processes in prokaryotic and eukaryotic organisms are often modulated by kinases which are in turn, dependent on Ca 2+ and the cyclic mononucleotides cAMP and cGMP. It has been established that some proteins have both kinase and cyclase activities and that active cyclases can be embedded within the kinase domains. Here, we identified phosphodiesterase (PDE) sites, enzymes that hydrolyse cAMP and cGMP, to AMP and GMP, respectively, in some of these proteins in addition to their kinase/cyclase twin-architecture. As an example, we tested the Arabidopsis thaliana KINγ, a subunit of the SnRK2 kinase, to demonstrate that all three enzymatic centres, adenylate cyclase (AC), guanylate cyclase (GC) and PDE, are catalytically active, capable of generating and hydrolysing cAMP and cGMP. These data imply that the signal output of the KINγ subunit modulates SnRK2, consequently affecting the downstream kinome. Finally, we propose a model where a single protein subunit, KINγ, is capable of regulating cyclic mononucleotide homeostasis, thereby tuning stimulus specific signal output., Competing Interests: Declaration of competing interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of the data; in the writing of the manuscript or in the decision to publish the results., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The polar auxin transport is required for proper plant growth and development. D6 PROTEIN KINASE (D6PK) is required for the phosphorylation of PIN-FORMED (PIN) auxin efflux carriers to regulate auxin transport, while the regulation of D6PK stabilization is still poorly understood. Here, we found that Cytosolic ABA Receptor Kinases (CARKs) redundantly interact with D6PK, and the interactions are dependent on CARKs' kinase activities. Similarly, CARK3 also could interact with paralogs of D6PK, including D6PKL1, D6PKL2, and D6PKL3. The genetic analysis shows that D6PK acts the downstream of CARKs to regulate Arabidopsis growth, including hypocotyl, leaf area, vein formation, and the length of silique. Loss-of-function of CARK3 in overexpressing GFP-D6PK plants leads to reduce the level of D6PK protein, thereby rescues plant growth. In addition, the cell-free degradation assays indicate that D6PK is degraded through 26 S proteasome pathway, while the phosphorylation by CARK3 represses this process in cells. In summary, D6PK stabilization by the CARK family is required for auxin-mediated plant growth and development., (© 2024 John Wiley & Sons Ltd.)

Phytohormone levels are regulated through specialized enzymes, participating not only in their biosynthesis but also in post-signaling processes for signal inactivation and cue depletion. Arabidopsis thaliana (At) carboxylesterase 15 (CXE15) and carboxylesterase 20 (CXE20) have been shown to deplete strigolactones (SLs) that coordinate various growth and developmental processes and function as signaling molecules in the rhizosphere. Here, we elucidate the X-ray crystal structures of AtCXE15 (both apo and SL intermediate bound) and AtCXE20, revealing insights into the mechanisms of SL binding and catabolism. The N-terminal regions of CXE15 and CXE20 exhibit distinct secondary structures, with CXE15 characterized by an alpha helix and CXE20 by an alpha/beta fold. These structural differences play pivotal roles in regulating variable SL hydrolysis rates. Our findings, both in vitro and in planta, indicate that a transition of the N-terminal helix domain of CXE15 between open and closed forms facilitates robust SL hydrolysis. The results not only illuminate the distinctive process of phytohormone breakdown but also uncover a molecular architecture and mode of plasticity within a specific class of carboxylesterases., (© 2024. The Author(s).)

In humans and plants, 40% of the proteome is cotranslationally acetylated at the N-terminus by a single Nα-acetyltransferase (Nat) termed NatA. The core NatA complex is comprised of the catalytic subunit Nα-acetyltransferase 10 (NAA10) and the ribosome-anchoring subunit NAA15. The regulatory subunit Huntingtin Yeast Partner K (HYPK) and the acetyltransferase NAA50 join this complex in humans. Even though both are conserved in Arabidopsis (Arabidopsis thaliana), only AtHYPK is known to interact with AtNatA. Here we uncover the AtNAA50 interactome and provide evidence for the association of AtNAA50 with NatA at ribosomes. In agreement with the latter, a split-luciferase approach demonstrated close proximity of AtNAA50 and AtNatA in planta. Despite their interaction, AtNatA/HYPK and AtNAA50 exerted different functions in vivo. Unlike NatA/HYPK, AtNAA50 did not modulate drought tolerance or promote protein stability. Instead, transcriptome and proteome analyses of a novel AtNAA50-depleted mutant (amiNAA50) implied that AtNAA50 negatively regulates plant immunity. Indeed, amiNAA50 plants exhibited enhanced resistance to oomycetes and bacterial pathogens. In contrast to what was observed in NatA-depleted mutants, this resistance was independent of an accumulation of salicylic acid prior to pathogen exposure. Our study dissects the in vivo function of the NatA interactors HYPK and NAA50 and uncovers NatA-independent roles for NAA50 in plants., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Prolyl 4-hydroxylase (P4H) generates hydroxyproline residues in proteins. Two classes of P4H have been found in plants. Type 1 P4H has a signal anchor at the N-terminus, while type 2 P4H has both an N-terminal signal peptide and a C-terminal toxin homology domain (Tox1 domain) with six conserved cysteine residues. We analyzed the localization of tobacco type 2 P4H (NtP4H2.2) in tobacco BY-2 cells. Cell fractionation studies, immunostaining of cells, and GFP fusion study indicated that NtP4H2.2 localizes predominantly to the Golgi apparatus and is a peripheral membrane protein associated with the luminal side of organelles. Expression of the GFP-Tox1 domains of NtP4H2.2 and another tobacco type 2 P4H NtP4H2.1 in BY-2 cells and Arabidopsis epidermal cells indicated that these proteins were targeted to the Golgi. The Tox1 domains from Arabidopsis and rice type 2 P4Hs also directed GFP to the Golgi in tobacco BY-2 cells. The Tox1 domain of NtP4H2.2 increased the membrane association of GFP, and mutation of the cysteine residues in this domain abolished Golgi localization. Furthermore, the catalytic domain of NtP4H2.2 also directed GFP to the Golgi. Thus, the Tox1 domains of plant P4Hs are the Golgi localization domains, and tobacco P4H2.2 localizes to the Golgi by the action of both this domain and the catalytic domain.

L-Arabinose (L-Ara) is a plant-specific sugar found in cell wall polysaccharides, proteoglycans, glycoproteins, and small glycoconjugates, which play physiologically important roles in cell proliferation and other essential cellular processes. L-Ara is synthesized as UDP-L-arabinose (UDP-L-Ara) from UDP-xylose (UDP-Xyl) by UDP-Xyl 4-epimerases (UXEs), a type of de novo synthesis of L-Ara unique to plants. In Arabidopsis, the Golgi-localized UXE AtMUR4 is the main contributor to UDP-L-Ara synthesis. However, cytosolic bifunctional UDP-glucose 4-epimerases (UGEs) with UXE activity, AtUGE1, and AtUGE3 also catalyze this reaction. For the present study, we first examined the physiological importance of bifunctional UGEs in Arabidopsis. The uge1 and uge3 mutants enhanced the dwarf phenotype of mur4 and further reduced the L-Ara content in cell walls, suggesting that bifunctional UGEs contribute to UDP-L-Ara synthesis. Through the introduction of point mutations exchanging corresponding amino acid residues between AtUGE1 with high UXE activity and AtUGE2 with low UXE activity, two mutations that increase relative UXE activity of AtUGE2 were identified. The crystal structures of AtUGE2 in complex forms with NAD + and NAD + /UDP revealed that the UDP-binding domain of AtUGE2 has a more closed conformation and smaller sugar-binding site than bacterial and mammalian UGEs, suggesting that plant UGEs have the appropriate size and shape for binding UDP-Xyl and UDP-L-Ara to exhibit UXE activity. The presented results suggest that the capacity for cytosolic synthesis of UDP-L-Ara was acquired by the small sugar-binding site and several mutations of UGEs, enabling diversified utilization of L-Ara in seed plants., (© 2024 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

The cuticle, consisting of cuticular wax and cutin, is a lipid membrane that seals the plant surface against environmental stress. β-Ketoacyl-CoA synthases (KCSs) are condensing enzymes catalyzing crucial reactions elongating hydrocarbon chains into precursors for various cuticular wax components. Although many KCS genes were well characterized in various species, the functions of the closely related Arabidopsis KCS3, KCS12, KCS19 enzymes remained unclear. Here, we found KCS3 preferentially expressed in growing organs, especially in guard cells. kcs3 mutants and kcs3kcs12 double mutants displayed sepal fusion phenotypes, suggesting defects in cuticle formation. The mutants had decreased amounts of wax components with relatively short hydrocarbon chains in the developing organs but increased levels of wax compounds in mature organs. In contrast, kcs19 mutants showed seed fusion phenotypes and altered chain length distributions in seed suberin. Taken together, our results show that KCS12 and KCS3 share redundant functions in flower development, while KCS19 is involved in seed coat formation. All three condensing enzymes are involved in the elongation of C >18 hydrocarbon chains in young, actively expanding tissues., (© 2024 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)