P-type calcium ATPases play important roles in biotic and abiotic stress signaling.

MAIN CONCLUSION: Knowledge of Ca2+-ATPases is imperative for improving crop quality/ food security, highly threatened due to global warming. Ca2+-ATPases modulates calcium, essential for stress signaling and modulating growth, development, and immune activities. Calcium is considered a versatile secondary messenger and essential for short- and long-term responses to biotic and abiotic stresses in plants. Coordinated transport activities from both calcium influx and efflux channels are required to generate cellular calcium signals. Various extracellular stimuli cause an induction in cytosolic calcium levels. To cope with such stresses, it is important to maintain intracellular Ca2+ levels. Plants need to evolve efficient efflux mechanisms to maintain Ca2+ ion homeostasis. Plant Ca2+-ATPases are members of the P-type ATPase superfamily and localized in the plasma membrane and endoplasmic reticulum (ER). They are required for various cellular processes, including plant growth, development, calcium signaling, and even retorts to environmental stress. These ATPases play an essential role in Ca2+ homeostasis and are actively involved in Ca2+ transport. Plant Ca2+-ATPases are categorized into two major classes: type IIA and type IIB. Although these two classes of ATPases share similarities in protein sequence, they differ in their structure, cellular localization, and sensitivity to inhibitors. Due to the emerging role of Ca2+-ATPase in abiotic and biotic plant stress, members of this family may help promote agricultural improvement under stress conditions. This review provides a comprehensive overview of P-type Ca2+-ATPase, and their role in Ca2+ transport, stress signaling, and cellular homeostasis focusing on their classification, evolution, ion specificities, and catalytic mechanisms. It also describes the main aspects of the role of Ca2+-ATPase in transducing signals during plant biotic and abiotic stress responses and its role in plant development and physiology.

Complete biosynthesis of QS-21 in engineered yeast.

QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans1,2. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis3,4. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host's native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families-a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases-from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure-activity relationship, and will thus enable the rational design of potent vaccine adjuvants.

Functions and mechanisms of enzymes assembling lignans and norlignans.

Lignans and norlignans are distributed throughout the plant kingdom and exhibit diverse chemical structures and biological properties that offer potential for therapeutic use. Originating from the phenylpropanoid biosynthesis pathway, their characteristic carbon architectures are formed through unique enzyme catalysis, featuring regio- and stereoselective C-C bond forming processes. Despite extensive research on these plant natural products, their biosynthetic pathways, and enzyme mechanisms remain enigmatic. This review highlights recent advancements in elucidating the functions and mechanisms of the biosynthetic enzymes responsible for constructing the distinct carbon frameworks of lignans and norlignans.

Mitochondrial RNA Helicases: Key Players in the Regulation of Plant Organellar RNA Splicing and Gene Expression.

Mitochondrial genomes of land plants are large and exhibit a complex mode of gene organization and expression, particularly at the post-transcriptional level. The primary organellar transcripts in plants undergo extensive maturation steps, including endo- and/or exo-nucleolytic cleavage, RNA-base modifications (mostly C-to-U deaminations) and both 'cis'- and 'trans'-splicing events. These essential processing steps rely on the activities of a large set of nuclear-encoded factors. RNA helicases serve as key players in RNA metabolism, participating in the regulation of transcription, mRNA processing and translation. They unwind RNA secondary structures and facilitate the formation of ribonucleoprotein complexes crucial for various stages of gene expression. Furthermore, RNA helicases are involved in RNA metabolism by modulating pre-mRNA maturation, transport and degradation processes. These enzymes are, therefore, pivotal in RNA quality-control mechanisms, ensuring the fidelity and efficiency of RNA processing and turnover in plant mitochondria. This review summarizes the significant roles played by helicases in regulating the highly dynamic processes of mitochondrial transcription, RNA processing and translation in plants. We further discuss recent advancements in understanding how dysregulation of mitochondrial RNA helicases affects the splicing of organellar genes, leading to respiratory dysfunctions, and consequently, altered growth, development and physiology of land plants.

Fatty acid de novo biosynthesis in plastids: Key enzymes and their critical roles for male reproduction and other processes in plants.

Fatty acid de novo biosynthesis in plant plastids is initiated from acetyl-CoA and catalyzed by a series of enzymes, which is required for the vegetative growth, reproductive growth, seed development, stress response, chloroplast development and other biological processes. In this review, we systematically summarized the fatty acid de novo biosynthesis-related genes/enzymes and their critical roles in various plant developmental processes. Based on bioinformatic analysis, we identified fatty acid synthase encoding genes and predicted their potential functions in maize growth and development, especially in anther and pollen development. Finally, we highlighted the potential applications of these fatty acid synthases in male-sterility hybrid breeding, seed oil content improvement, herbicide and abiotic stress resistance, which provides new insights into future molecular crop breeding.

Small ubiquitin-like modifiers E3 ligases in plant stress.

Plants regularly encounter various environmental stresses such as salt, drought, cold, heat, heavy metals and pathogens, leading to changes in their proteome. Of these, a post-translational modification, SUMOylation is particularly significant for its extensive involvement in regulating various plant molecular processes to counteract these external stressors. Small ubiquitin-like modifiers (SUMO) protein modification significantly contributes to various plant functions, encompassing growth, development and response to environmental stresses. The SUMO system has a limited number of ligases even in fully sequenced plant genomes but SUMO E3 ligases are pivotal in recognising substrates during the process of SUMOylation. E3 ligases play pivotal roles in numerous biological and developmental processes in plants, including DNA repair, photomorphogenesis, phytohormone signalling and responses to abiotic and biotic stress. A considerable number of targets for E3 ligases are proteins implicated in reactions to abiotic and biotic stressors. This review sheds light on how plants respond to environmental stresses by focusing on recent findings on the role of SUMO E3 ligases, contributing to a better understanding of how plants react at a molecular level to such stressors.

Differential Substrate Sensing in Terpene Synthases from Plants and Microorganisms: Insight from Structural, Bioinformatic, and EnzyDock Analyses.

Terpene synthases (TPSs) catalyze the first step in the formation of terpenoids, which comprise the largest class of natural products in nature. TPSs employ a family of universal natural substrates, composed of isoprenoid units bound to a diphosphate moiety. The intricate structures generated by TPSs are the result of substrate binding and folding in the active site, enzyme-controlled carbocation reaction cascades, and final reaction quenching. A key unaddressed question in class I TPSs is the asymmetric nature of the diphosphate-(Mg2+)3 cluster, which forms a critical part of the active site. In this asymmetric ion cluster, two diphosphate oxygen atoms protrude into the active site pocket. The substrate hydrocarbon tail, which is eventually molded into terpenes, can bind to either of these oxygen atoms, yet to which is unknown. Herein, we employ structural, bioinformatics, and EnzyDock docking tools to address this enigma. We bring initial data suggesting that this difference is rooted in evolutionary differences between TPSs. We hypothesize that this alteration in binding, and subsequent chemistry, is due to TPSs originating from plants or microorganisms. We further suggest that this difference can cast light on the frequent observation that the chiral products or intermediates of plant and bacterial terpene synthases represent opposite enantiomers.

Physiological function and regulation of ascorbate peroxidase isoforms.

Ascorbate peroxidase (APX) reduces H2O2 to H2O by utilizing ascorbate as a specific electron donor and constitutes the ascorbate-glutathione cycle in organelles of plants including chloroplasts, cytosol, mitochondria, and peroxisomes. It has been almost 40 years since APX was discovered as an important plant-specific H2O2-scavenging enzyme, during which time many research groups have conducted molecular physiological analyses. It is now clear that APX isoforms function not only just as antioxidant enzymes but also as important factors in intracellular redox regulation through the metabolism of reactive oxygen species. The function of APX isoforms is regulated at multiple steps, from the transcriptional level to post-translational modifications of enzymes, thereby allowing them to respond flexibly to ever-changing environmental factors and physiological phenomena such as cell growth and signal transduction. In this review, we summarize the physiological functions and regulation mechanisms of expression of each APX isoform.

Revisiting the role of ascorbate oxidase in plant systems.

Ascorbic acid (AsA) plays an indispensable role in plants, serving as both an antioxidant and a master regulator of the cellular redox balance. Ascorbate oxidase (AO) is a blue copper oxidase that is responsible for the oxidation of AsA with the concomitant production of water. For many decades, AO was erroneously postulated as an enzyme without any obvious advantage, as it decreases the AsA pool size and thus is expected to weaken plant stress resistance. It was only a decade ago that this perspective shifted towards the fundamental role of AO in orchestrating both AsA and oxygen levels by influencing the overall redox balance in the extracellular matrix. Consistent with its localization in the apoplast, AO is involved in cell expansion, division, resource allocation, and overall plant yield. An increasing number of transgenic studies has demonstrated that AO can also facilitate communication between the surrounding environment and the cell, as its gene expression is highly responsive to factors such as hormonal signaling, oxidative stress, and mechanical injury. This review aims to describe the multiple functions of AO in plant growth, development, and stress resilience, and explore any additional roles the enzyme might have in fruits during the course of ripening.

Cysteine thiol sulfinic acid in plant stress signaling.

Cysteine thiols are susceptible to various oxidative posttranslational modifications (PTMs) due to their high chemical reactivity. Thiol-based PTMs play a crucial role in regulating protein functions and are key contributors to cellular redox signaling. Although reversible thiol-based PTMs, such as disulfide bond formation, S-nitrosylation, and S-glutathionylation, have been extensively studied for their roles in redox regulation, thiol sulfinic acid (-SO2H) modification is often perceived as irreversible and of marginal significance in redox signaling. Here, we revisit this narrow perspective and shed light on the redox regulatory roles of -SO2H in plant stress signaling. We provide an overview of protein sulfinylation in plants, delving into the roles of hydrogen peroxide-mediated and plant cysteine oxidase-catalyzed formation of -SO2H, highlighting the involvement of -SO2H in specific regulatory signaling pathways. Additionally, we compile the existing knowledge of the -SO2H reducing enzyme, sulfiredoxin, offering insights into its molecular mechanisms and biological relevance. We further summarize current proteomic techniques for detecting -SO2H and furnish a list of experimentally validated cysteine -SO2H sites across various species, discussing their functional consequences. This review aims to spark new insights and discussions that lead to further investigations into the functional significance of protein -SO2H-based redox signaling in plants.

Photosystem II Subunit S (PsbS): A Nano Regulator of Plant Photosynthesis.

Light is required for photosynthesis, but plants are often exposed to excess light, which can lead to photodamage and eventually cell death. To prevent this, they evolved photoprotective feedback mechanisms that regulate photosynthesis and trigger processes that dissipate light energy as heat, called non-photochemical quenching (NPQ). In excess light conditions, the light reaction and activity of Photosystem II (PSII) generates acidification of the thylakoid lumen, which is sensed by special pH-sensitive proteins called Photosystem II Subunit S (PsbS), actuating a photoprotective "switch" in the light-harvesting antenna. Despite its central role in regulating photosynthetic energy conversion, the molecular mechanism of PsbS as well as its interaction with partner proteins are not well understood. This review summarizes the current knowledge on the molecular structure and mechanistic aspects of the light-stress sensor PsbS and addresses open questions and challenges in the field regarding a full understanding of its functional mechanism and role in NPQ.

Plant carbonic anhydrase-like enzymes in neuroactive alkaloid biosynthesis.

Plants synthesize numerous alkaloids that mimic animal neurotransmitters1. The diversity of alkaloid structures is achieved through the generation and tailoring of unique carbon scaffolds2,3, yet many neuroactive alkaloids belong to a scaffold class for which no biosynthetic route or enzyme catalyst is known. By studying highly coordinated, tissue-specific gene expression in plants that produce neuroactive Lycopodium alkaloids4, we identified an unexpected enzyme class for alkaloid biosynthesis: neofunctionalized α-carbonic anhydrases (CAHs). We show that three CAH-like (CAL) proteins are required in the biosynthetic route to a key precursor of the Lycopodium alkaloids by catalysing a stereospecific Mannich-like condensation and subsequent bicyclic scaffold generation. Also, we describe a series of scaffold tailoring steps that generate the optimized acetylcholinesterase inhibition activity of huperzine A5. Our findings suggest a broader involvement of CAH-like enzymes in specialized metabolism and demonstrate how successive scaffold tailoring can drive potency against a neurological protein target.

A tale of two switches: Redox regulation of adenosine-5'-phosphosulfate kinase in humans and plants.

The sulfate donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is a near-universal component of sulfur metabolism. In a report by Zhang et al. in this issue of Structure, X-ray crystal structures of the APS kinase domains from human PAPS synthase reveal dynamic substrate recognition and a regulatory "redox switch" analogous to that previously described only in plant APS kinases.

Recent Advances of Polyphenol Oxidases in Plants.

Polyphenol oxidase (PPO) is present in most higher plants, but also in animals and fungi. PPO in plants had been summarized several years ago. However, recent advances in studies of PPO in plants are lacking. This review concludes new researches on PPO distribution, structure, molecular weights, optimal temperature, pH, and substrates. And, the transformation of PPO from latent to active state was also discussed. This state shift is a vital reason for elevating PPO activity, but the activation mechanism in plants has not been elucidated. PPO has an important role in plant stress resistance and physiological metabolism. However, the enzymatic browning reaction induced by PPO is a major problem in the production, processing, and storage of fruits and vegetables. Meanwhile, we summarized various new methods that had been invented to decrease enzymatic browning by inhibiting PPO activity. In addition, our manuscript included information on several important biological functions and the transcriptional regulation of PPO in plants. Furthermore, we also prospect some future research areas of PPO and hope they will be useful for future research in plants.

A higher plant FAD synthetase is fused to an inactivated FAD pyrophosphatase.

The riboflavin derivatives FMN and flavin adenine dinucleotide (FAD) are critical cofactors for wide-ranging biological processes across all kingdoms of life. Although it is well established that these flavins can be readily interconverted, in plants, the responsible catalysts and regulatory mechanisms remain poorly understood. Here, we report the cloning and biochemical characterization of an FAD synthetase encoded by the gene At5g03430, which we have designated AtFADS1 (A. thaliana FADS1). The catalytic properties of the FAD synthetase activity are similar to those reported for other FAD synthetases, except that we observed maximum activity with Zn2+ as the associated divalent metal cation. Like human FAD synthetase, AtFADS1 exists as an apparent fusion with an ancestral FAD pyrophosphatase, a feature that is conserved across plants. However, we detected no pyrophosphatase activity with AtFADS1, consistent with an observed loss of a key catalytic residue in higher plant evolutionary history. In contrast, we determined that algal FADS1 retains both FAD synthetase and pyrophosphatase activity. We discuss the implications, including the potential for yet-unstudied biologically relevant noncatalytic functions, and possible evolutionary pressures that have led to the loss of FAD pyrophosphatase activity, yet universal retention of an apparently nonfunctional domain in FADS of land plants.

Dissipation of the proton electrochemical gradient in chloroplasts promotes the oxidation of ATP synthase by thioredoxin-like proteins.

Chloroplast FoF1-ATP synthase (CFoCF1) uses an electrochemical gradient of protons across the thylakoid membrane (ΔµH+) as an energy source in the ATP synthesis reaction. CFoCF1 activity is regulated by the redox state of a Cys pair on its central axis, that is, the γ subunit (CF1-γ). When the ΔµH+ is formed by the photosynthetic electron transfer chain under light conditions, CF1-γ is reduced by thioredoxin (Trx), and the entire CFoCF1 enzyme is activated. The redox regulation of CFoCF1 is a key mechanism underlying the control of ATP synthesis under light conditions. In contrast, the oxidative deactivation process involving CFoCF1 has not been clarified. In the present study, we analyzed the oxidation of CF1-γ by two physiological oxidants in the chloroplast, namely the proteins Trx-like 2 and atypical Cys-His-rich Trx. Using the thylakoid membrane containing the reduced form of CFoCF1, we were able to assess the CF1-γ oxidation ability of these Trx-like proteins. Our kinetic analysis indicated that these proteins oxidized CF1-γ with a higher efficiency than that achieved by a chemical oxidant and typical chloroplast Trxs. Additionally, the CF1-γ oxidation rate due to Trx-like proteins and the affinity between them were changed markedly when ΔµH+ formation across the thylakoid membrane was manipulated artificially. Collectively, these results indicate that the formation status of the ΔµH+ controls the redox regulation of CFoCF1 to prevent energetic disadvantages in plants.

HECT ubiquitin ligases as accessory proteins of the plant proteasome.

The proteasome plays vital roles in eukaryotic cells by orchestrating the regulated degradation of large repertoires of substrates involved in numerous biological processes. Proteasome dysfunction is associated with a wide variety of human pathologies and in plants severely affects growth, development and responses to stress. The activity of E3 ubiquitin ligases marks proteins fated for degradation with chains of the post-translational modifier, ubiquitin. Proteasomal processing of ubiquitinated substrates involves ubiquitin chain recognition, deubiquitination, ATP-mediated unfolding and translocation, and proteolytic digestion. This complex series of steps is made possible not only by the many specialised subunits of the 1.5 MDa proteasome complex but also by a range of accessory proteins that are recruited to the proteasome. A surprising class of accessory proteins are members of the HECT-type family of ubiquitin ligases that utilise a unique mechanism for post-translational attachment of ubiquitin to their substrates. So why do proteasomes that already contain all the necessary machinery to recognise ubiquitinated substrates, harbour HECT ligase activity? It is now clear that some ubiquitin ligases physically relay their substrates to proteasome-associated HECT ligases, which prevent substrate stalling at the proteasome. Moreover, HECT ligases ubiquitinate proteasome subunits, thereby modifying the proteasome's ability to recognise substrates. They may therefore enable proteasomes to be both non-specific and extraordinarily selective in a complex substrate environment. Understanding the relationship between the proteasome and accessory HECT ligases will reveal how the proteasome controls so many diverse plant developmental and stress responses.

Heme oxygenase-nitric oxide crosstalk-mediated iron homeostasis in plants under oxidative stress.

Plant growth under abiotic stress conditions significantly enhances intracellular generation of reactive oxygen species (ROS). Oxidative status of plant cells is directly affected by the modulation of iron homeostasis. Among mammals and plants, heme oxygenase-1 (HO-1) is a well-known antioxidant enzyme. It catalyzes oxygenation of heme, thereby producing Fe2+, CO and biliverdin as byproducts. The antioxidant potential of HO-1 is primarily due to its catalytic reaction byproducts. Biliverdin and bilirubin possess conjugated π-electrons which escalate the ability of these biomolecules to scavenge free radicals. CO also enhances the ROS scavenging ability of plants cells by upregulating catalase and peroxidase activity. Enhanced expression of HO-1 in plants under oxidative stress accompanies sequestration of iron in specialized iron storage proteins localized in plastids and mitochondria, namely ferritin for Fe3+ storage and frataxin for storage of Fe-S clusters, respectively. Nitric oxide (NO) crosstalks with HO-1 at multiple levels, more so in plants under oxidative stress, in order to maintain intracellular iron status. Formation of dinitrosyl-iron complexes (DNICs) significantly prevents Fenton reaction during oxidative stress. DNICs also release NO upon dissociation in target cells over long distance in plants. They also function as antioxidants against superoxide anions and lipidic free radicals. A number of NO-modulated transcription factors also facilitate iron homeostasis in plant cells. Plants facing oxidative stress exhibit modulation of lateral root formation by HO-1 through NO and auxin-dependent pathways. The present review provides an in-depth analysis of the structure-function relationship of HO-1 in plants and mammals, correlating them with their adaptive mechanisms of survival under stress.