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In bacteria and archaea, proteins of the ParA/MinD family of ATPases regulate the spatiotemporal organization of various cellular cargoes, including cell division proteins, motility structures, chemotaxis systems, and chromosomes. In bacteria, such as Escherichia coli, MinD proteins are crucial for the correct placement of the Z-ring at mid-cell during cell division. However, previous studies have shown that none of the 4 MinD homologs present in the archaeon Haloferax volcanii have a role in cell division, suggesting that these proteins regulate different cellular processes in haloarchaea. Here, we show that while deletion of MinD2 in H. volcanii (∆minD2) does not affect cell growth or division, it impacts cell shape and motility by mispositioning the chemotaxis arrays and archaellum motors. Finally, we explore the links between MinD2 and MinD4, which has been previously shown to modulate the localization of chemosensory arrays and archaella in H. volcanii, finding that the two MinD homologues have synergistic effects in regulating the positioning of the motility machinery. Collectively, our findings identify MinD2 as an important link between cell shape and motility in H. volcanii and further our understanding of the mechanisms by which multiple MinD proteins regulate cellular functions in haloarchaea.

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.

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.

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.

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.

The vacuole has a space-filling function, allowing a particularly rapid plant cell expansion with very little increase in cytosolic content (Löfke et al., 2015; Scheuring et al., 2016; Dünser et al., 2019). Despite its importance for cell size determination in plants, very little is known about the mechanisms that define vacuolar size. Here, we show that the cellular and vacuolar size expansions are coordinated. By developing a pharmacological tool, we enabled the investigation of membrane delivery to the vacuole during cellular expansion. Our data reveal that endocytic membrane sorting from the plasma membrane to the vacuole is enhanced in the course of rapid root cell expansion. While this ‘compromise’ mechanism may theoretically at first decelerate cell surface enlargements, it fuels vacuolar expansion and, thereby, ensures the coordinated augmentation of vacuolar occupancy in dynamically expanding plant cells.

Hypersaline environments are the source of many viruses infecting different species of halophilic euryarchaea. Information on infection mechanisms of archaeal viruses is scarce, due to the lack of genetically accessible virus–host models. Recently, a new archaeal siphovirus, Haloferax tailed virus 1 (HFTV1), was isolated together with its host belonging to the genus Haloferax, but it is not infectious on the widely used model euryarcheon Haloferax volcanii. To gain more insight into the biology of HFTV1 host strain LR2-5, we studied characteristics that might play a role in its virus susceptibility: growth-dependent motility, surface layer, filamentous surface structures, and cell shape. Its genome sequence showed that LR2-5 is a new strain of Haloferax gibbonsii. LR2-5 lacks obvious viral defense systems, such as CRISPR-Cas, and the composition of its cell surface is different from Hfx. volcanii, which might explain the different viral host range. This work provides first deep insights into the relationship between the host of halovirus HFTV1 and other members of the genus Haloferax. Given the close relationship to the genetically accessible Hfx. volcanii, LR2-5 has high potential as a new model for virus–host studies in euryarchaea.

ABSTRACT 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.

Several haloarchaea are reported to be pleomorphic, while others exhibit remarkable shapes, such as squares. Recently, Haloferax volcanii was found to alter its morphology during growth. Cells are motile rods in early exponential phase, and immotile plates in stationary phase. It is unknown if this growth phase dependent cell shape alteration is a specific feature of Hfx. volcanii, or conserved amongst haloarchaea. Here, we studied the cell shape and motility of two haloarchaea species Haloarcula hispanica and Haloarcula californiae. With a combination of light and electron microscopy, we observed that both strains undergo a growth phase dependent morphological development, albeit in a slightly different fashion as Hfx. volcanii. For both Haloarcula strains, the cell size is changing throughout growth. Cell shape seems to be related with motility, as highly motile cells on semi-solid agar plates are predominantly rod-shaped. We conclude that the growth phase dependent cell morphology alteration might be a common feature amongst haloarchaea, and that cell shape is generally linked with a motile life style. The conservation of this phenomenon underscores the importance of studies of the molecular mechanisms regulating cell shape in archaea.

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.

Recombinant phytoene desaturase (PDS-His6) from rice was purified to near-homogeneity and shown to be enzymatically active in a biphasic, liposome-based assay system. The protein contains FAD as the sole protein-bound redox-cofactor. Benzoquinones, not replaceable by molecular oxygen, serve as a final electron acceptor defining PDS as a 15-cis-phytoene (donor):plastoquinone oxidoreductase. The herbicidal PDS-inhibitor norflurazon is capable of arresting the reaction by stabilizing the intermediary FAD(red), while an excess of the quinone acceptor relieves this blockage, indicating competition. The enzyme requires its homo-oligomeric association for activity. The sum of data collected through gel permeation chromatography, non-denaturing polyacrylamide electrophoresis, chemical cross-linking, mass spectrometry and electron microscopy techniques indicate that the high-order oligomers formed in solution are the basis for an active preparation. Of these, a tetramer consisting of dimers represents the active unit. This is corroborated by our preliminary X-ray structural analysis that also revealed similarities of the protein fold with the sequence-inhomologous bacterial phytoene desaturase CRTI and other oxidoreductases of the GR2-family of flavoproteins. This points to an evolutionary relatedness of CRTI and PDS yielding different carotene desaturation sequences based on homologous protein folds.