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Nitrogen-fixing nodulation occurs in 10 taxonomic lineages, with either rhizobia or Frankia bacteria. To establish such an endosymbiosis, two processes are essential: nodule organogenesis and intracellular bacterial infection. In the legume–rhizobium endosymbiosis, both processes are guarded by the transcription factor NODULE INCEPTION (NIN) and its downstream target genes of the NUCLEAR FACTOR Y (NF-Y) complex. It is hypothesized that nodulation has a single evolutionary origin c. 110 Ma, followed by many independent losses. Despite a significant body of knowledge of the legume–rhizobium symbiosis, it remains elusive which signalling modules are shared between nodulating species in different taxonomic clades. We used Parasponia andersonii to investigate the role of NIN and NF-YA genes in rhizobium nodulation in a nonlegume system. Consistent with legumes, P. andersonii PanNIN and PanNF-YA1 are coexpressed in nodules. By analyzing single, double and higher-order CRISPR-Cas9 knockout mutants, we show that nodule organogenesis and early symbiotic expression of PanNF-YA1 are PanNIN-dependent and that PanNF-YA1 is specifically required for intracellular rhizobium infection. This demonstrates that NIN and NF-YA1 have conserved symbiotic functions. As Parasponia and legumes diverged soon after the birth of the nodulation trait, we argue that NIN and NF-YA1 represent core transcriptional regulators in this symbiosis.

The importance of the arginyl‐tRNA protein transferase (ATE), the enzyme mediating post‐translation arginylation of proteins in the N‐end rule degradation (NERD) pathway of protein stability, was analysed in Physcomitrella patens and compared to its known functions in other eukaryotes. We characterize ATE:GUS reporter lines as well as ATE mutants in P. patens to study the impact and function of arginylation on moss development and physiology. ATE protein abundance is spatially and temporally regulated in P. patens by hormones and light and is highly abundant in meristematic cells. Further, the amount of ATE transcript is regulated during abscisic acid signalling and downstream of auxin signalling. Loss‐of‐function mutants exhibit defects at various levels, most severely in developing gametophores, in chloroplast starch accumulation and senescence. Thus, arginylation is necessary for moss gametophyte development, in contrast to the situation in flowering plants. Our analysis further substantiates the conservation of the N‐end rule pathway components in land plants and highlights lineage‐specific features. We introduce moss as a model system to characterize the role of the NERD pathway as an additional layer of complexity in eukaryotic development.

Reactive oxygen species (ROS) are constant by-products of aerobic life. In excess, ROS lead to cytotoxic protein aggregates, which are a hallmark of ageing in animals and linked to age-related pathologies in humans. Acylamino acid-releasing enzymes (AARE) are bifunctional serine proteases, acting on oxidized proteins. AARE are found in all domains of life, albeit under different names, such as acylpeptide hydrolase (APEH/ACPH), acylaminoacyl peptidase (AAP), or oxidized protein hydrolase (OPH). In humans, AARE malfunction is associated with age-related pathologies, while their function in plants is less clear. Here, we provide a detailed analysis of AARE genes in the plant lineage and an in-depth analysis of AARE localization and function in the moss Physcomitrella and the angiosperm Arabidopsis. AARE loss-of-function mutants have not been described for any organism so far. We generated and analysed such mutants and describe a connection between AARE function, aggregation of oxidized proteins and plant ageing, including accelerated developmental progression and reduced life span. Our findings complement similar findings in animals and humans, and suggest a unified concept of ageing may exist in different life forms. The analysis of the function of acylamino acid-releasing enzymes (AARE) in Physcomitrella and Arabidopsis reveals a connection between AARE and plant ageing, suggesting a unified concept of ageing may exist across domains of life.

The net amounts of carotenoids accumulating in plant tissues are determined by the rates of biosynthesis and degradation. While biosynthesis is rate-limited by the activity of PHYTOENE SYNTHASE (PSY), carotenoid losses are caused by catabolic enzymatic and non-enzymatic degradation. We established a system based on non-green Arabidopsis callus which allowed investigating major determinants for high steady-state levels of β-carotene. Wild-type callus development was characterized by strong carotenoid degradation which was only marginally caused by the activity of carotenoid cleavage oxygenases. In contrast, carotenoid degradation occurred mostly non-enzymatically and selectively affected carotenoids in a molecule-dependent manner. Using carotenogenic pathway mutants, we found that linear carotenes such as phytoene, phytofluene and pro-lycopene resisted degradation and accumulated while β-carotene was highly susceptible towards degradation. Moderately increased pathway activity through PSY overexpression was compensated by degradation revealing no net increase in β-carotene. However, higher pathway activities outcompeted carotenoid degradation and efficiently increased steady-state β-carotene amounts to up to 500 μg g-1 dry mass. Furthermore, we identified oxidative β-carotene degradation products which correlated with pathway activities, yielding β-apocarotenals of different chain length and various apocarotene-dialdehydes. The latter included methylglyoxal and glyoxal as putative oxidative end products suggesting a potential recovery of carotenoid-derived carbon for primary metabolic pathways. Moreover, we investigated the site of β-carotene sequestration by co-localization experiments which revealed that β-carotene accumulated as intra-plastid crystals which was confirmed by electron microscopy with carotenoid-accumulating roots. The results are discussed in the context of using the non-green calli carotenoid assay system for approaches targeting high steady-state β-carotene levels prior to their application in crops.

Arabidopsis (Arabidopsis thaliana) SALT TOLERANCE/B-BOX ZINC FINGER PROTEIN24 (STO/BBX24) is a negative regulator of the light signal transduction that localizes to the nucleus of plant cells and interacts with CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) in the yeast (Saccharomyces cerevisiae) two-hybrid system. The protein contains two B-box zinc-finger motives at the N terminus and a conserved motif at the C-terminal part required for the interaction with COP1. BBX24 accumulates during deetiolation of young seedlings in the first hours of exposure to light. However, this accumulation is transient and decreases after prolonged light irradiation. Here, we identified the amino acidic residues necessary for the nuclear import of the protein. In addition, we created mutated forms of the protein, and analyzed them by overexpression in the bbx24-1 mutant background. Our results indicate that the degradation of BBX24 occurs, or at least is initiated in the nucleus, and this nuclear localization is a prerequisite to fulfill its function in light signaling. Moreover, mutations in the region responsible for the interaction with COP1 revealed that a physical interaction of the proteins is also required for degradation of BBX24 in the light and for normal photomorphogenesis.

The extracellular matrix plays an integrative role in cellular responses in plants, but its contribution to the signalling of extracellular ligands largely remains to be explored. Rapid alkalinisation factors (RALFs) are extracellular peptide hormones that play pivotal roles in various physiological processes. Here, we address a crucial connection between the de-methylesterification machinery of the cell wall component pectin and RALF1 activity. Pectin is a polysaccharide, contributing to the structural integrity of the cell wall. Our data illustrate that the pharmacological and genetic interference with pectin methyl esterases (PMEs) abolishes RALF1-induced root growth repression. Our data suggest that positively charged RALF1 peptides bind negatively charged, de-methylesterified pectin with high avidity. We illustrate that the RALF1 association with de-methylesterified pectin is required for its FERONIA-dependent perception, contributing to the control of the extracellular matrix and the regulation of plasma membrane dynamics. Notably, this mode of action is independent of the FER-dependent extracellular matrix sensing mechanism provided by FER interaction with the leucine-rich repeat extensin (LRX) proteins. We propose that the methylation status of pectin acts as a contextualizing signalling scaffold for RALF peptides, linking extracellular matrix dynamics to peptide hormone-mediated responses.

Host-controlled intracellular accommodation of nitrogen-fixing bacteria is essential for the establishment of a functional Root Nodule Symbiosis (RNS). In many host plants, this occurs via transcellular tubular structures (infection threads - ITs) that extend across cell layers via polar tip-growth. Comparative phylogenomic studies have identified RPG ( RHIZOBIUM-DIRECTED POLAR GROWTH ) among the critical genetic determinants for bacterial infection. In Medicago truncatula , RPG is required for effective IT progression within root hairs but the cellular and molecular function of the encoded protein remains elusive. Here, we show that RPG resides in the protein complex formed by the core endosymbiotic components VAPYRIN (VPY) and LUMPY INFECTION (LIN) required for IT polar growth, co-localizes with both VPY and LIN in IT tip- and perinuclear-associated puncta of M. truncatula root hairs undergoing infection and is necessary for VPY recruitment into these structures. Fluorescence Lifetime Imaging Microscopy (FLIM) of phosphoinositide species during bacterial infection revealed that functional RPG is required to sustain strong membrane polarization at the advancing tip of the IT. In addition, loss of RPG functionality alters the cytoskeleton-mediated connectivity between the IT tip and the nucleus and affects the polar secretion of the cell wall modifying enzyme NODULE PECTATE LYASE (NPL). Our results integrate RPG into a core host machinery required to support symbiont accommodation, suggesting that its occurrence in plant host genomes is essential to co-opt a multimeric protein module committed to endosymbiosis to sustain IT-mediated bacterial infection.

In plants, the topological organization of membranes has mainly been attributed to the cell wall and the cytoskeleton. Additionally, few proteins, such as plant-specific remorins have been shown to function as protein and lipid organizers. Root nodule symbiosis requires continuous membrane re-arrangements, with bacteria being finally released from infection threads into membrane-confined symbiosomes. We found that mutations in the symbiosis-specific SYMREM1 gene result in highly disorganized perimicrobial membranes. AlphaFold modelling and biochemical analyses reveal that SYMREM1 oligomerizes into antiparallel dimers and may form a higher-order membrane scaffolding structure. This was experimentally confirmed when expressing this and other remorins in wall-less protoplasts is sufficient where they significantly alter and stabilize de novo membrane topologies ranging from membrane blebs to long membrane tubes with a central actin filament. Reciprocally, mechanically induced membrane indentations were equally stabilized by SYMREM1. Taken together we describe a plant-specific mechanism that allows the stabilization of large-scale membrane conformations independent of the cell wall. In plants, plasma membrane topologies are predominantly driven by the cell wall. In this study, the authors demonstrate that remorin proteins can take over these functions at specialized, unwalled plasma membranes such as infection droplets associated with symbiotic infection threads.

Motility is a central feature of many microorganisms and provides an efficient strategy to respond to environmental changes. Bacteria and archaea have developed fundamentally different rotary motors enabling their motility, termed flagellum and archaellum, respectively. Bacterial motility along chemical gradients, called chemotaxis, critically relies on the response regulator CheY, which, when phosphorylated, inverses the rotational direction of the flagellum via a switch complex at the base of the motor. The structural difference between archaellum and flagellum and the presence of functional CheY in archaea raises the question of how the CheY protein changed to allow communication with the archaeal motility machinery. Here we show that archaeal CheY shares the overall structure and mechanism of magnesium-dependent phosphorylation with its bacterial counterpart. However, bacterial and archaeal CheY differ in the electrostatic potential of the helix α4. The helix α4 is important in bacteria for interaction with the flagellar switch complex, a structure that is absent in archaea. We demonstrated that phosphorylation-dependent activation, and conserved residues in the archaeal CheY helix α4, are important for interaction with the archaeal-specific adaptor protein CheF. This forms a bridge between the chemotaxis system and the archaeal motility machinery. Conclusively, archaeal CheY proteins conserved the central mechanistic features between bacteria and archaea, but differ in the helix α4 to allow binding to an archaellum-specific interaction partner.

Archaea are ubiquitous single cellular microorganisms that play important ecological roles in nature. The intracellular organization of archaeal cells is among the unresolved mysteries of archaeal biology. With this work, we show that cells of haloarchaea are polarized. The cellular positioning of proteins involved in chemotaxis and motility is spatially and temporally organized in these cells. This suggests the presence of a specific mechanism responsible for the positioning of macromolecular protein complexes in archaea. Bacteria and archaea exhibit tactical behavior and can move up and down chemical gradients. This tactical behavior relies on a motility structure, which is guided by a chemosensory system. Environmental signals are sensed by membrane-inserted chemosensory receptors that are organized in large ordered arrays. While the cellular positioning of the chemotaxis machinery and that of the flagellum have been studied in detail in bacteria, we have little knowledge about the localization of such macromolecular assemblies in archaea. Although the archaeal motility structure, the archaellum, is fundamentally different from the flagellum, archaea have received the chemosensory machinery from bacteria and have connected this system with the archaellum. Here, we applied a combination of time-lapse imaging and fluorescence and electron microscopy using the model euryarchaeon Haloferax volcanii and found that archaella were specifically present at the cell poles of actively dividing rod-shaped cells. The chemosensory arrays also had a polar preference, but in addition, several smaller arrays moved freely in the lateral membranes. In the stationary phase, rod-shaped cells became round and chemosensory arrays were disassembled. The positioning of archaella and that of chemosensory arrays are not interdependent and likely require an independent form of positioning machinery. This work showed that, in the rod-shaped haloarchaeal cells, the positioning of the archaellum and of the chemosensory arrays is regulated in time and in space. These insights into the cellular organization of H. volcanii suggest the presence of an active mechanism responsible for the positioning of macromolecular protein complexes in archaea. IMPORTANCE Archaea are ubiquitous single cellular microorganisms that play important ecological roles in nature. The intracellular organization of archaeal cells is among the unresolved mysteries of archaeal biology. With this work, we show that cells of haloarchaea are polarized. The cellular positioning of proteins involved in chemotaxis and motility is spatially and temporally organized in these cells. This suggests the presence of a specific mechanism responsible for the positioning of macromolecular protein complexes in archaea.