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Publikation

Abukhalaf, M.; Proksch, C.; Thieme, D.; Ziegler, J.; Hoehenwarter, W.; Changing turn-over rates regulate abundance of tryptophan, GS biosynthesis, IAA transport and photosynthesis proteins in Arabidopsis growth defense transitions BMC Biol. 21, 249, (2023) DOI: 10.1186/s12915-023-01739-3

Background Shifts in dynamic equilibria of the abundance of cellular molecules in plant-pathogen interactions need further exploration. We induced PTI in optimally growing Arabidopsis thaliana seedlings for 16 h, returning them to growth conditions for another 16 h. Methods Turn-over and abundance of 99 flg22 responding proteins were measured chronologically using a stable heavy nitrogen isotope partial labeling strategy and targeted liquid chromatography coupled to mass spectrometry (PRM LC–MS). These experiments were complemented by measurements of mRNA and phytohormone levels. Results Changes in synthesis and degradation rate constants (Ks and Kd) regulated tryptophane and glucosinolate, IAA transport, and photosynthesis-associated protein (PAP) homeostasis in growth/PTI transitions independently of mRNA levels. Ks values increased after elicitation while protein and mRNA levels became uncorrelated. mRNA returned to pre-elicitation levels, yet protein abundance remained at PTI levels even 16 h after media exchange, indicating protein levels were robust and unresponsive to transition back to growth. The abundance of 23 PAPs including FERREDOXIN-NADP( +)-OXIDOREDUCTASE (FNR1) decreased 16 h after PAMP exposure, their depletion was nearly abolished in the myc234 mutant. FNR1 Kd increased as mRNA levels decreased early in PTI, its Ks decreased in prolonged PTI. FNR1 Kd was lower in myc234, mRNA levels decreased as in wild type. Conclusions Protein Kd and Ks values change in response to flg22 exposure and constitute an additional layer of protein abundance regulation in growth defense transitions next to changes in mRNA levels. Our results suggest photosystem remodeling in PTI to direct electron flow away from the photosynthetic carbon reaction towards ROS production as an active defense mechanism controlled post-transcriptionally and by MYC2 and homologs. Target proteins accumulated later and PAP and auxin/IAA depletion was repressed in myc234 indicating a positive effect of the transcription factors in the establishment of PTI.
Publikation

Nietzschmann, L.; Smolka, U.; Perino, E. H. B.; Gorzolka, K.; Stamm, G.; Marillonnet, S.; Bürstenbinder, K.; Rosahl, S.; The secreted PAMP-induced peptide StPIP1_1 activates immune responses in potato Sci. Rep. 13, 20534, (2023) DOI: 10.1038/s41598-023-47648-x

Treatment of potato plants with the pathogen-associated molecular pattern Pep-13 leads to the activation of more than 1200 genes. One of these, StPIP1_1, encodes a protein of 76 amino acids with sequence homology to PAMP-induced secreted peptides (PIPs) from Arabidopsis thaliana. Expression of StPIP1_1 is also induced in response to infection with Phytophthora infestans, the causal agent of late blight disease. Apoplastic localization of StPIP1_1-mCherry fusion proteins is dependent on the presence of the predicted signal peptide. A synthetic peptide corresponding to the last 13 amino acids of StPIP1_1 elicits the expression of the StPIP1_1 gene itself, as well as that of pathogenesis related genes. The oxidative burst induced by exogenously applied StPIP1_1 peptide in potato leaf disks is dependent on functional StSERK3A/B, suggesting that StPIP1_1 perception occurs via a receptor complex involving the co-receptor StSERK3A/B. Moreover, StPIP1_1 induces expression of FRK1 in Arabidopsis in an RLK7-dependent manner. Expression of an RLK from potato with high sequence homology to AtRLK7 is induced by StPIP1_1, by Pep-13 and in response to infection with P. infestans. These observations are consistent with the hypothesis that, upon secretion, StPIP1_1 acts as an endogenous peptide required for amplification of the defense response.
Publikation

Yang, B.; Stamm, G.; Bürstenbinder, K.; Voiniciuc, C.; Microtubule‐associated IQD9 orchestrates cellulose patterning in seed mucilage New Phytol. 235, 1096-1110, (2022) DOI: 10.1111/nph.18188

Arabidopsis seeds release large capsules of mucilaginous polysaccharides, which are shaped by an intricate network of cellulosic microfibrils. Cellulose synthase complexes are guided by the microtubule cytoskeleton, but it is unclear which proteins mediate this process in the seed coat epidermis. 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 in cell wall polysaccharide biosynthesis. Mutations in IQD9 as well as in KLCR1 lead to compact mucilage capsules with aberrant cellulose distribution, which can be rescued by transgene complementation. IQD9 physically interacts with KLCR1 and localizes to cortical MTs to maintain their organization in SCE cells. IQD9 as well as a previously identified TONNEAU1 (TON1) RECRUITING MOTIF 4 (TRM4) protein act to maintain cellulose synthase velocity. 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 cell wall biosynthesis. Therefore, SCE cells provide an attractive system to further decipher the complex genetic regulation of polarized cellulose deposition.
Publikation

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.
Publikation

Zang, J.; Klemm, S.; Pain, C.; Duckney, P.; Bao, Z.; Stamm, G.; Kriechbaumer, V.; Bürstenbinder, K.; Hussey, P. J.; Wang, P.; A novel plant actin-microtubule bridging complex regulates cytoskeletal and ER structure at ER-PM contact sites Curr. Biol. 31, 1251-1260, (2021) DOI: 10.1016/j.cub.2020.12.009

In plants, the cortical endoplasmic reticulum (ER) network is connected to the plasma membrane (PM) through the ER-PM contact sites (EPCSs), whose structures are maintained by EPCS resident proteins and the cytoskeleton.1-7 Strong co-alignment between EPCSs and the cytoskeleton is observed in plants,1,8 but little is known of how the cytoskeleton is maintained and regulated at the EPCS. Here, we have used a yeast-two-hybrid screen and subsequent in vivo interaction studies in plants by fluorescence resonance energy transfer (FRET)-fluorescence lifetime imaging microscopy (FLIM) analysis to identify two microtubule binding proteins, KLCR1 (kinesin-light-chain-related protein 1) and IQD2 (IQ67-domain 2), that interact with the actin binding protein NET3C and form a component of plant EPCS that mediates the link between the actin and microtubule networks. The NET3C-KLCR1-IQD2 module, acting as an actin-microtubule bridging complex, has a direct influence on ER morphology and EPCS structure. Their loss-of-function mutants, net3a/NET3C RNAi, klcr1, or iqd2, exhibit defects in pavement cell morphology, which we suggest is linked to the disorganization of both actin filaments and microtubules. In conclusion, our results reveal a novel cytoskeletal-associated complex, which is essential for the maintenance and organization of cytoskeletal structure and ER morphology at the EPCS and for normal plant cell morphogenesis.
Publikation

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.
Preprints

Zang, J.; Klemm, S.; Pain, C.; Duckney, P.; Bao, Z.; Stamm, G.; Kriechbaumer, V.; Bürstenbinder, K.; Hussey, P. J.; Wang, P.; A Novel Plant Actin-Microtubule Bridging Complex Regulates Cytoskeletal and ER Structure at Endoplasmic Reticulum-Plasma Membrane Contact Sites (EPCS) SSRN Electronic Journal (2020) DOI: 10.2139/ssrn.3581370

In plants, the cortical ER network is connected to the plasma membrane through the ER-PM contact sites (EPCS), whose structures are maintained by EPCS resident proteins and the cytoskeleton. Strong co-alignment between EPCS and the cytoskeleton is observed in plants, but little is known of how the cytoskeleton is maintained and regulated at the EPCS. Here we have used a yeast-two-hybrid screen and subsequent in vivo interaction studies in plants by FRET-FLIM analysis, to identify two microtubule binding proteins, KLCR1 (Kinesin Light Chain Related protein 1) and IQD2 (IQ67-Domain 2) that interact with the actin binding protein NET3C and form a component of plant EPCS, that mediates the link between the actin and microtubule networks. The NET3C-KLCR1-IQD2 module, acting as an actin-microtubule bridging complex, has a direct influence on ER morphology. Their loss of function mutants, net3a/NET3C RNAi, 0klcr1 or iqd2, exhibit defects in pavement cell morphology which we suggest is linked to the disorganization of both actin filaments and microtubules. In conclusion, our results reveal a novel cytoskeletal associated complex, which is essential for the maintenance and organization of both cytoskeletal structure and ER morphology at the EPCS, and for normal plant cell morphogenesis.
Preprints

Bassal, M.; Majovsky, P.; Thieme, D.; Herr, T.; Abukhalaf, M.; Ayash, M.; Al Shweiki, M. R.; Proksch, C.; Hmedat, A.; Ziegler, J.; Neumann, S.; Hoehenwarter, W.; Reshaping of the Arabidopsis thaliana proteome landscape and co-regulation of proteins in development and immunity bioRxiv (2020) DOI: 10.1101/2020.03.09.978627

Proteome remodeling is a fundamental adaptive response and proteins in complex and functionally related proteins are often co-expressed. Using a deep sampling strategy we define Arabidopsis thaliana tissue core proteomes at around 10,000 proteins per tissue and absolutely quantify (copy numbers per cell) nearly 16,000 proteins throughout the plant lifecycle. A proteome wide survey of global post translational modification revealed amino acid exchanges pointing to potential conservation of translational infidelity in eukaryotes. Correlation analysis of protein abundance uncovered potentially new tissue and age specific roles of entire signaling modules regulating transcription in photosynthesis, seed development and senescence and abscission. Among others, the data suggest a potential function of RD26 and other NAC transcription factors in seed development related to desiccation tolerance as well as a possible function of Cysteine-rich Receptor-like Kinases (CRKs) as ROS sensors in senescence. All of the components of ribosome biogenesis factor (RBF) complexes were co-expressed tissue and age specifically indicating functional promiscuity in the assembly of these little described protein complexes in Arabidopsis. Treatment of seedlings with flg22 for 16 hours allowed us to characterize proteome architecture in basal immunity in detail. The results were complemented with parallel reaction monitoring (PRM) targeted proteomics, phytohormone, amino acid and transcript measurements. We obtained strong evidence of suppression of jasmonate (JA) and JA-Ile levels by deconjugation and hydroxylation via IAA-ALA RESISTANT3 (IAR3) and JASMONATE-INDUCED OXYGENASE 2 (JOX2) under the control of JASMONATE INSENSITIVE 1 (MYC2). This previously unknown regulatory switch is another part of the puzzle of the as yet understudied role of JA in pattern triggered immunity. The extensive coverage of the Arabidopsis proteome in various biological scenarios presents a rich resource to plant biologists that we make available to the community.
Publikation

Niemeyer, M.; Moreno Castillo, E.; Ihling, C. H.; Iacobucci, C.; Wilde, V.; Hellmuth, A.; Hoehenwarter, W.; Samodelov, S. L.; Zurbriggen, M. D.; Kastritis, P. L.; Sinz, A.; Calderón Villalobos, L. I. A.; Flexibility of intrinsically disordered degrons in AUX/IAA proteins reinforces auxin co-receptor assemblies Nat. Commun. 11, 2277, (2020) DOI: 10.1038/s41467-020-16147-2

Cullin RING-type E3 ubiquitin ligases SCFTIR1/AFB1-5 and their AUX/IAA targets perceive the phytohormone auxin. The F-box protein TIR1 binds a surface-exposed degron in AUX/IAAs promoting their ubiquitylation and rapid auxin-regulated proteasomal degradation. Here, by adopting biochemical, structural proteomics and in vivo approaches we unveil how flexibility in AUX/IAAs and regions in TIR1 affect their conformational ensemble allowing surface accessibility of degrons. We resolve TIR1·auxin·IAA7 and TIR1·auxin·IAA12 complex topology, and show that flexible intrinsically disordered regions (IDRs) in the degron’s vicinity, cooperatively position AUX/IAAs on TIR1. We identify essential residues at the TIR1 N- and C-termini, which provide non-native interaction interfaces with IDRs and the folded PB1 domain of AUX/IAAs. We thereby establish a role for IDRs in modulating auxin receptor assemblies. By securing AUX/IAAs on two opposite surfaces of TIR1, IDR diversity supports locally tailored positioning for targeted ubiquitylation, and might provide conformational flexibility for a multiplicity of functional states.
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