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Publications - Molecular Signal Processing

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Publications

Prasad, A.; Breithaupt, C.; Nguyen, D.-A.; Lilie, H.; Ziegler, J.; Stubbs, M. T.; Mechanism of chorismate dehydratase MqnA, the first enzyme of the futalosine pathway, proceeds via substrate-assisted catalysis J. Biol. Chem. 298, 102601, (2022) DOI: 10.1016/j.jbc.2022.102601

MqnA, the only chorismate dehydratase known so far, catalyzes the initial step in the biosynthesis of menaquinone via the futalosine pathway. Details of the MqnA reaction mechanism remain unclear. Here, we present crystal structures of Streptomyces coelicolor MqnA and its active site mutants in complex with chorismate and the product 3-enolpyruvyl-benzoate, produced during heterologous expression in Escherichia coli. Together with activity studies, our data are in line with dehydration proceeding via substrate assisted catalysis, with the enol pyruvyl group of chorismate acting as catalytic base. Surprisingly, structures of the mutant Asn17Asp with copurified ligand suggest that the enzyme converts to a hydrolase by serendipitous positioning of the carboxyl group. All complex structures presented here exhibit a closed Venus flytrap fold, with the enzyme exploiting the characteristic ligand binding properties of the fold for specific substrate binding and catalysis. The conformational rearrangements that facilitate complete burial of substrate/product, with accompanying topological changes to the enzyme surface, could foster substrate channeling within the biosynthetic pathway.
Publications

Naumann, C.; Heisters, M.; Brandt, W.; Janitza, P.; Alfs, C.; Tang, N.; Toto Nienguesso, A.; Ziegler, J.; Imre, R.; Mechtler, K.; Dagdas, Y.; Hoehenwarter, W.; Sawers, G.; Quint, M.; Abel, S.; Bacterial-type ferroxidase tunes iron-dependent phosphate sensing during Arabidopsis root development Curr. Biol. 32, 2189-2205, (2022) DOI: 10.1016/j.cub.2022.04.005

Access to inorganic phosphate (Pi), a principal intermediate of energy and nucleotide metabolism, profoundly affects cellular activities and plant performance. In most soils, antagonistic Pi-metal interactions restrict Pi bioavailability, which guides local root development to maximize Pi interception. Growing root tips scout the essential but immobile mineral nutrient; however, the mechanisms monitoring external Pi sta-tus are unknown. Here, we show that Arabidopsis LOW PHOSPHATE ROOT 1 (LPR1), one key determinant of Fe-dependent Pi sensing in root meristems, encodes a novel ferroxidase of high substrate specificity and affinity (apparent KM ∼2 μmM Fe2+). LPR1 typifies an ancient, Fe-oxidizing multicopper protein family that evolved early upon bacterial land colonization. The ancestor of streptophyte algae and embryophytes (land plants) acquired LPR1-type ferroxidase from soil bacteria via horizontal gene transfer, a hypothesis supported by phylogenomics, homology modeling, and biochemistry. Our molecular and kinetic data on LPR1 regulation indicate that Pi-dependent Fe substrate availability determines LPR1 activity and function. Guided by the metabolic lifestyle of extant sister bacterial genera, we propose that Arabidopsis LPR1 monitors subtle concentration differentials of external Fe availability as a Pi-dependent cue to adjust root meristem maintenance via Fe redox signaling and cell wall modification. We further hypothesize that the acquisition of bacterial LPR1-type ferroxidase by embryophyte progenitors facilitated the evolution of local Pi sensing and acquisition during plant terrestrialization.
Preprints

Yang, B.; Stamm, G.; Bürstenbinder, K.; Voiniciuc, C.; Microtubule-associated IQD9 guides cellulose synthase velocity to shape seed mucilage bioRxiv (2021) DOI: 10.1101/2021.12.11.472226

SummaryArabidopsis seeds release large capsules of mucilaginous polysaccharides, which are shaped by an intricate network of cellulosic microfibrils. Cellulose synthase complexes is guided by the microtubule cytoskeleton, but it is unclear which proteins mediate this process in the seed coat epidermis (SCE).Using reverse genetics, we identified IQ67 DOMAIN 9 (IQD9) and KINESIN LIGHT CHAIN-RELATED 1 (KLCR1) as two highly expressed genes during seed development and comprehensively characterized their roles for cell wall polysaccharide biosynthesis and cortical microtubule (MT) organization.Mutations in IQD9 as well as in KLCR1 lead to compact mucilage capsules with aberrant cellulose distribution, which can be rescued by transgene complementation. Double mutant analyses revealed that their closest paralogs (IQD10 and KLCR2, respectively) are not required for mucilage biosynthesis. IQD9 physically interacts with KLCR1 and localizes to cortical MTs to maintain their organization in SCE cells. Similar to the previously identified TONNEAU1 (TON1) RECRUITING MOTIF 4 (TRM4) protein, IQD9 is required to maintain the velocity of cellulose synthases.Our results demonstrate that IQD9, KLCR1 and TRM4 are MT-associated proteins that are required for seed mucilage architecture. This study provides the first direct evidence that members of the IQD, KLCR and TRM families have overlapping roles in guiding the distribution of cell wall polysaccharides. Therefore, SCE cells provide an attractive system to further decipher the complex genetic regulation of polarized cellulose deposition.
Publications

Mielke, S.; Zimmer, M.; Meena, M. K.; Dreos, R.; Stellmach, H.; Hause, B.; Voiniciuc, C.; Gasperini, D.; Jasmonate biosynthesis arising from altered cell walls is prompted by turgor-driven mechanical compression Sci. Adv. 7, eabf0356, (2021) DOI: 10.1126/sciadv.abf0356

Despite the vital roles of jasmonoyl-isoleucine (JA-Ile) in governing plant growth and environmental acclimation, it remains unclear what intracellular processes lead to its induction. Here, we provide compelling genetic evidence that mechanical and osmotic regulation of turgor pressure represents a key elicitor of JA-Ile biosynthesis. After identifying cell wall mutant alleles in KORRIGAN1 (KOR1) with elevated JA-Ile in seedling roots, we found that ectopic JA-Ile resulted from cell nonautonomous signals deriving from enlarged cortex cells compressing inner tissues and stimulating JA-Ile production. Restoring cortex cell size by cell type–specific KOR1 complementation, by isolating a genetic kor1 suppressor, and by lowering turgor pressure with hyperosmotic treatments abolished JA-Ile signaling. Conversely, hypoosmotic treatment activated JA-Ile signaling in wild-type plants. Furthermore, constitutive JA-Ile levels guided mutant roots toward greater water availability. Collectively, these findings enhance our understanding on JA-Ile biosynthesis initiation and reveal a previously undescribed role of JA-Ile in orchestrating environmental resilience.
Publications

Mercier, C.; Roux, B.; Havé, M.; Le Poder, L.; Duong, N.; David, P.; Leonhardt, N.; Blanchard, L.; Naumann, C.; Abel, S.; Cuyas, L.; Pluchon, S.; Laurent, N.; Desnos, T.; Root responses to aluminium and iron stresses require the SIZ1 SUMO ligase to modulate the STOP1 transcription factor Plant J. 108, 1507-1521, (2021) DOI: 10.1111/tpj.15525

STOP1, an Arabidopsis transcription factor favouring root growth tolerance against Al toxicity, acts in the response to iron under low Pi (-Pi). Previous studies have shown that Al and Fe regulate the stability and accumulation of STOP1 in roots, and that the STOP1 protein is sumoylated by an unknown E3 ligase. Here, using a forward genetics suppressor screen, we identified the E3 SUMO (small ubiquitin-like modifier) ligase SIZ1 as a modulator of STOP1 signalling. Mutations in SIZ1 increase the expression of ALMT1 (a direct target of STOP1) and root growth responses to Al and Fe stress in a STOP1-dependent manner. Moreover, loss-of-function mutations in SIZ1 enhance the abundance of STOP1 in the root tip. However, no sumoylated STOP1 protein was detected by western blot analysis in our sumoylation assay in E. coli, suggesting the presence of a more sophisticated mechanism. We conclude that the sumo ligase SIZ1 negatively regulates STOP1 signalling, at least in part by modulating STOP1 protein in the root tip. Our results will allow a better understanding of this signalling pathway.
Publications

Menna, A.; Dora, S.; Sancho-Andrés, G.; Kashyap, A.; Meena, M. K.; Sklodowski, K.; Gasperini, D.; Coll, N. S.; Sánchez-Rodríguez, C.; A primary cell wall cellulose-dependent defense mechanism against vascular pathogens revealed by time-resolved dual transcriptomics BMC Biol. 19, 161, (2021) DOI: 10.1186/s12915-021-01100-6

Background: Cell walls (CWs) are protein-rich polysaccharide matrices essential for plant growth and environmental acclimation. The CW constitutes the first physical barrier as well as a primary source of nutrients for microbes interacting with plants, such as the vascular pathogen Fusarium oxysporum (Fo). Fo colonizes roots, advancing through the plant primary CWs towards the vasculature, where it grows causing devastation in many crops. The pathogenicity of Fo and other vascular microbes relies on their capacity to reach and colonize the xylem. However, little is known about the root-microbe interaction before the pathogen reaches the vasculature and the role of the plant CW during this process. Results: Using the pathosystem Arabidopsis-Fo5176, we show dynamic transcriptional changes in both fungus and root during their interaction. One of the earliest plant responses to Fo5176 was the downregulation of primary CW synthesis genes. We observed enhanced resistance to Fo5176 in Arabidopsis mutants impaired in primary CW cellulose synthesis. We confirmed that Arabidopsis roots deposit lignin in response to Fo5176 infection, but we show that lignin-deficient mutants were as susceptible as wildtype plants to Fo5176. Genetic impairment of jasmonic acid biosynthesis and signaling did not alter Arabidopsis response to Fo5176, whereas impairment of ethylene signaling did increase vasculature colonization by Fo5176. Abolishing ethylene signaling attenuated the observed resistance while maintaining the dwarfism observed in primary CW cellulose-deficient mutants. Conclusions: Our study provides significant insights on the dynamic root-vascular pathogen interaction at the transcriptome level and the vital role of primary CW cellulose during defense response to these pathogens. These findings represent an essential resource for the generation of plant resistance to Fo that can be transferred to other vascular pathosystems.Keywords: Arabidopsis, Fusarium oxysporum, Ralstonia solanacearum, plant-pathogen interaction, dual-time coursetranscriptomics, cellulose, ethylene, defense response
Publications

Meena, S.; Wagner, C.; Caggegi, L.; Baumann-Kaschig, K.; Ried, M. K.; A user-friendly protocol for the cultivation and successful crossing of Lotus japonicus Bio Protoc. (2021) DOI: 10.21769/p1464

This is a detailed and user-friendly protocol for the cultivation and successful crossing of Lotus japonicus (L. japonicus) e.g. for the generation of higher order mutants, based on methods previously reported (Grant et al., 1962; Handberg and Stougaards, 1992; Jiang and Gresshoff, 1997; Pajuelo and Stougaard, 2005).
Publications

Kumari, P.; Dahiya, P.; Livanos, P.; Zergiebel, L.; Kölling, M.; Poeschl, Y.; Stamm, G.; Hermann, A.; Abel, S.; Müller, S.; Bürstenbinder, K.; IQ67 DOMAIN proteins facilitate preprophase band formation and division-plane orientation Nat. Plants 7, 739-747, (2021) DOI: 10.1038/s41477-021-00923-z

Spatiotemporal control of cell division is essential for the growth and development of multicellular organisms. In plant cells, proper cell plate insertion during cytokinesis relies on the premitotic establishment of the division plane at the cell cortex. Two plant-specific cytoskeleton arrays, the preprophase band (PPB) and the phragmoplast, play important roles in division-plane orientation and cell plate formation, respectively1. Microtubule organization and dynamics and their communication with membranes at the cortex and cell plate are coordinated by multiple, mostly distinct microtubule-associated proteins2. How division-plane selection and establishment are linked, however, is still unknown. Here, we report members of the Arabidopsis IQ67 DOMAIN (IQD) family3 as microtubule-targeted proteins that localize to the PPB and phragmoplast and additionally reside at the cell plate and a polarized cortical region including the cortical division zone (CDZ). IQDs physically interact with PHRAGMOPLAST ORIENTING KINESIN (POK) proteins4,5 and PLECKSTRIN HOMOLOGY GTPase ACTIVATING (PHGAP) proteins6, which are core components of the CDZ1. The loss of IQD function impairs PPB formation and affects CDZ recruitment of POKs and PHGAPs, resulting in division-plane positioning defects. We propose that IQDs act as cellular scaffolds that facilitate PPB formation and CDZ set-up during symmetric cell division.
Publications

Klionsky, D. J.; Abdel-Aziz, A. K.; Abdelfatah, S.; Abdellatif, M.; Abdoli, A.; Abel, S.; Naumann, C.; et al., .; Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition) Autophagy 17, 1-382, (2021) DOI: 10.1080/15548627.2020.1797280

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis  updated  guidelines  for  monitoring  autophagy  in  different  organisms.  Despite  numerous  reviews, there  continues  to  be  confusion  regarding  acceptable  methods  to  evaluate  autophagy,  especially  in multicellular  eukaryotes.  Here,  we  present  a  set  of  guidelines  for  investigators  to  select  and  interpret methods  to  examine  autophagy  and  related  processes,  and  for  reviewers  to  provide  realistic  and reasonable  critiques  of  reports  that  are  focused  on  these  processes.  These  guidelines  are  not  meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being  asked  and  the  system  being  used.  Moreover,  no  individual  assay  is  perfect  for  every situation, calling  for  the  use  of  multiple  techniques  to  properly  monitor  autophagy  in  each  experimental  setting. Finally,  several  core  components  of  the  autophagy  machinery  have  been  implicated  in  distinct  auto-phagic  processes  (canonical  and  noncanonical  autophagy),  implying  that  genetic  approaches  to  block autophagy  should  rely  on  targeting  two  or  more  autophagy-related  genes  that  ideally  participate  in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation  in  the  field.
Publications

Kahsay, B. N.; Ziegler, J.; Imming, P.; Gebre-Mariam, T.; Neubert, R. H. H.; Moeller, L.; Free amino acid contents of selected Ethiopian plant and fungi species: a search for alternative natural free amino acid sources for cosmeceutical applications Amino Acids 53, 1105-1122, (2021) DOI: 10.1007/s00726-021-03008-5

Free amino acids (FAAs), the major constituents of the natural moisturizing factor (NMF), are very important for maintaining the moisture balance of human skin and their deficiency results in dry skin conditions. There is a great interest in the identification and use of nature-based sources of these molecules for such cosmeceutical applications. The objective of the present study was, therefore, to investigate the FAA contents of selected Ethiopian plant and fungi species; and select the best sources so as to use them for the stated purpose. About 59 different plant species and oyster mushroom were included in the study and the concentrations of 27 FAAs were analyzed. Each sample was collected, lyophilized, extracted using aqueous solvent, derivatized with Fluorenylmethoxycarbonyl chloride (Fmoc-Cl) prior to solid-phase extraction and quantified using Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC-ESI–MS/MS) system. All the 27 FAAs were detected in most of the samples. The dominant FAAs that are part of the NMF were found at sufficiently high concentration in the mushroom and some of the plants. This indicates that FAAs that could be included in the preparations for the management of dry skin condition can be obtained from a single natural resource and the use of these resources for the specified purpose have both economic and therapeutic advantage in addition to fulfilling customer needs.
  • Die Leibniz-Gemeinschaft
  • Digitalization in agriculture
  • Universität Halle
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