Biology subjects

Schaaf, G.

Publications and source records attributed to Schaaf, G..

16 recordsLinked to original sources

bioRxiv2025

Systems-level Plant Responses Reveal Pseudomonas-Mediated Growth Promotion in Brachypodium Under Nitrogen Limitation

Plant molecular adaptation to plant growth promoting bacteria (PGPB) under nutrient stress remains unclear, yet is essential for advancing PGPB use in agriculture. The model grass Brachypodium dystachion was studied together with Pseudomonas koreeensis (Pk) at two nitrogen (N) conditions. Non-invasive shoot phenotyping showed an immediate response to low-N, while beneficial effects of Pk became quantifiable after day 19. Increased N content in inoculated plants, along with Pks ability to grow on N-free media, suggests bacterial N contribution at deficient N. In low-N conditions, Pk-inoculated plants showed 33.2% more N than uninoculated controls and biomass comparable to high-N plants. Pk had no effect under sufficient N. Proteomics and lipidomics revealed that lipid profiles were primarily shaped by N availability, while protein abundance responded to both Pk and N status. Inoculated low-N plants displayed protein profiles resembling those of high-N controls, with some distinct exceptions. The plant-microbe interaction is dynamic and developed over 3 weeks, leading to increased biomass and N content. Root proteins strongly induced by Pk under low-N included lipid degradation enzymes, N transporters, and regulatory proteins, suggesting a coordinated remodelling of energy metabolism supporting whole-plant biomass and increased abundance of N uptake proteins.

plant biology↗

bioRxiv2025

Affinity-Based Interactome Mapping of Inositol Pyrophosphates Reveals 4/6-PP-InsP5-Binding Proteins in Plants

Inositol pyrophosphates (PP-InsPs) are central regulators of eukaryotic signaling events. While certain PP-InsP isomers have been conclusively linked to the regulation of phosphate homeostasis through interaction with SPX domain containing proteins in plants, the functions of the recently discovered isomer 4/6-PP-InsP5 remain largely unknown. Here, we employed two complementary affinity-based strategies - a matrix approach and a photoaffinity probe - to systematically identify 4/6-PP-InsP5-binding proteins in Arabidopsis thaliana. The two methods yielded partially overlapping protein sets, with photoaffinity enrichment likely capturing additional transient and/or weak interactions. Moreover, competition experiments with different isomers were applied to obtain information about potential isomer-specific interactions. As a proof-of-concept, one candidate interactor (FHA domain-containing protein AtFHA2) was shown to bind 4-PP-InsP5 in vitro with substantially higher affinity than InsP6. Thus, besides the SPX domain, FHA domain containing proteins, of which 18 exist in Arabidopsis, are potentially regulated by inositol pyrophosphates. More generally, our findings reveal a diverse protein network associated with 4/6-PP-InsP5 and establish a versatile platform for dissecting its biological roles in plants and other organisms.

biochemistry↗

bioRxiv2024

Lotus japonicus VIH2 is an inositol pyrophosphate synthase that regulates arbuscular mycorrhiza

Plant yield is often maximized by the extensive use of mineral fertilizers, which, however, has severe environmental consequences. Phosphate is particularly problematic, as it represents a globally limited resource, and its runoff and soil erosion threaten open water bodies. Many crops engage in arbuscular mycorrhizal (AM) symbiosis with nutrient-acquiring fungi, aiding in the uptake of phosphate and other mineral nutrients. However, AM colonization is strongly reduced under high soil phosphate levels. A mechanistic understanding of phosphate sensing, phosphate starvation responses, and their connection to AM remains enigmatic. Here, we show that in Lotus japonicus, low-abundant, energy-rich inositol pyrophosphates act as master regulators of AM, orchestrating the crosstalk between phosphate starvation responses and plant root endosymbiosis. These findings hold promise for breeding nutrient-efficient crops.

plant biology↗

bioRxiv2024

Association of the benzoxazinoid pathway with boron homeostasis in maize

Both deficiency and toxicity of the micronutrient boron lead to severe reductions in crop yield. Despite this agricultural importance, the molecular basis underlying boron homeostasis in plants is not resolved. To identify molecular players involved in boron homeostasis in maize (Zea mays), we measured boron levels in the Goodman-Buckler association panel and performed genome-wide association studies. These analyses detected the benzoxazinless (bx) gene, bx3, involved in the biosynthesis of benzoxazinoids, like DIMBOA, major defense compounds in maize. Genes involved in DIMBOA biosynthesis are all located in close proximity in the genome and the biosynthesis mutants, including bx3 are all DIMBOA deficient. We show that the bx3 mutant has enhanced boron concentration in leaves compared to the B73 control plants, which correlates with enhanced leaf tip necrosis, a phenotype associated with boron toxicity. In contrast, other DIMBOA-deficient maize mutants did not show altered boron levels nor the leaf tip necrosis phenotype, suggesting that boron is not associated with DIMBOA. Instead, our analyses suggest that the accumulation of boron is linked to the benzoxazinoid intermediates indolin-2-one (ION) and 3-hydroxy-ION. Therefore, our results connect boron homeostasis to the benzoxazinoid plant defense pathway through bx3 and specific intermediates, rendering the benzoxazinoid biosynthesis pathway a potential target for crop improvement in inadequate boron conditions. One sentence summaryGWAS identified benzoxazinless3 in the benzoxazinoid biosynthesis pathway to play a role in boron homeostasis in maize.

plant biology↗

bioRxiv2024

NUDIX Hydrolases Target Specific Inositol Pyrophosphates and Regulate Phosphate and Iron Homeostasis, and the Expression of Defense Genes in Arabidopsis

Inositol pyrophosphates (PP-InsPs) are important signaling molecules that regulate diverse cellular processes in eukaryotes, including energy homeostasis, phosphate (Pi) signaling, and phytohormone perception. Yet, in plants, the enzymes responsible for their turnover remain largely unknown. Using a non-hydrolysable PP-InsP analog in a pull-down approach, we identified a family of Arabidopsis NUDIX hydrolases (NUDTs) that group into two closely related subclades. Through in vitro assays, heterologous expression systems, and higher-order gene-edited mutants, we explored the substrate specificities and physiological roles of these hydrolases. Using a combination of strong anion exchange (SAX)-HPLC, PAGE, and capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS), we found that their PP-InsP pyrophosphatase activity is enantiomer-selective and Mg2+-dependent. Specifically, subclade I NUDTs preferentially hydrolyze 4-InsP7, while subclade II NUDTs target 3-InsP7, with minor activity against other PP-InsPs, including 5-InsP7. In higher-order mutants of subclade II NUDTs, we observed defects in both Pi and iron homeostasis, accompanied by increased levels of 1/3-InsP7 and 5-InsP7, with a markedly larger increase in 1/3-InsP7. Ectopic expression of NUDTs from both subclades induced local Pi starvation responses (PSRs), while RNA-seq analysis comparing wildtype (WT) and subclade II nudt12/13/16 loss-of-function plants indicates additional PSR-independent roles, potentially involving 1/3-InsP7 in the regulation of plant defense. Consistently, nudt12/13/16 mutants displayed enhanced resistance to Pseudomonas syringae infection, indicating a role in bacterial pathogen susceptibility. Expanding beyond subclade II NUDTs, we demonstrated susceptibility of the 3PP-position of PP-InsPs to enzymatic activities unrelated to NUDTs, and found that such activities are conserved across plants and humans. Additionally, we found that NUDT effectors from pathogenic ascomycete fungi exhibit a substrate specificity similar to subclade I NUDTs. Collectively, our findings reveal new roles for NUDTs in PP-InsP signaling, plant nutrient and immune responses, and highlight a cross-kingdom conservation of PP-InsP-metabolizing enzymes.

plant biology↗

bioRxiv2023

Bacterial Pathogen Infection Triggers Magic Spot Nucleotide Signaling in Arabidopsis thaliana Chloroplasts Through Specific RelA/SpoT Homologs

Magic spot nucleotides (p)ppGpp are important signaling molecules in bacteria and plants. In the latter, RSH enzymes are responsible for (p)ppGpp turnover. Profiling of (p)ppGpp is more difficult in plants than in bacteria due to lower concentrations and more severe matrix effects. Here, we report that capillary electrophoresis mass spectrometry (CE-MS) can be deployed to study (p)ppGpp abundance and identity in Arabidopsis thaliana. This goal is achieved by combining a titanium dioxide extraction protocol and pre-spiking with chemically synthesized stable isotope labeled internal reference compounds. The high sensitivity and separation efficiency of CE-MS enables monitoring of changes in (p)ppGpp levels in A. thaliana upon infection with the pathogen Pseudomonas syringae pv. tomato (PstDC3000). We observed a significant increase of ppGpp post infection that is also stimulated by the flagellin peptide flg22 only. This increase depends on functional flg22 receptor FLS2 and its interacting kinase BAK1 indicating that pathogen-associated molecular pattern (PAMP) receptor-mediated signaling controls ppGpp levels. Transcript analyses showed an upregulation of RSH2 upon flg22 treatment and both RSH2 and RSH3 after PstDC3000 infection. A. thaliana mutants deficient in RSH2 and RSH3 activity display no ppGpp accumulation upon infection and flg22 treatment, supporting involvement of these synthases in PAMP-triggered innate immunity responses to pathogens within the chloroplast.

biochemistry↗

bioRxiv2023

Heritable maize microbiomes contribute to local adaptation and host stress resilience

Beneficial interactions with microorganisms are pivotal for crop performance and resilience. However, it remains unclear how heritable the microbiome is with respect to the host plant genotype and to what extent host genetic mechanisms can modulate plant-microbe interactions in the face of environmental stress. Here, we surveyed the root and rhizosphere microbiome of 129 accessions of locally adapted Zea mays, sourced from diverse habitats and grown under control and different stress conditions. We quantified treatment and host genotype effects on the microbiome. Plant genotype and source environment were predictive of microbiome composition. Genome wide association analysis identified host genetic variants linked to both rhizosphere microbiome composition and source environment. We identified transposon insertions in a candidate gene linked to both the abundance of a keystone microbe Massilia and source total soil nitrogen, finding mutant plants to show a reduction in lateral root density. We conclude that locally adapted maize varieties exert patterns of genetic control on their root and rhizosphere microbiomes that follow variation in their home environments, consistent with a role in tolerance to prevailing stress.

genetics↗

bioRxiv2022

Mechanisms by Which Small Molecule Inhibitors Arrest Sec14 Phosphatidylinositol Transfer Protein Activity

Phosphatidylinositol transfer proteins (PITPs) promote phosphoinositide signaling by enhancing phosphatidylinositol (PtdIns) 4-OH kinase activities in producing signaling pools of PtdIns-4-phosphate. As such, PITPs are key regulators of lipid signaling in eukaryotic cells. While the PITP phospholipid exchange cycle is the engine that stimulates PtdIns 4-OH kinase activity, the protein and lipid dynamics associated with this critical process are not understood. Herein, we use an integrative structural approach that takes advantage of small molecule inhibitors (SMIs) directed against the major yeast PITP (Sec14) to gain new insights into the mechanics of the Sec14 phospholipid exchange cycle from the perspective of protein, phospholipid and SMI dynamics. Moreover, as Sec14 has emerged as an attractive target for next-generation antifungal drugs, the structures of Sec14 bound to SMIs of four different chemotypes reported in this study provide critical information required for structure-based design of next-generation lead compounds that target Sec14 PITPs of virulent fungi.

biophysics↗

bioRxiv2022

Phase Separation of a Nodulin Sec14-like protein Maintains Auxin Efflux Carrier Polarity at Arabidopsis Plasma Membranes

Protein function can be modulated by phase transitions in their material properties, which can range from liquid-to solid-like; yet the mechanisms that drive these transitions and whether they are important for physiology are still unknown. Using the model plant Arabidopsis, we show that developmental robustness is reinforced by phase transitions of the plasma membrane-bound lipid-binding protein SEC14-like. Using imaging, genetics, and in vitro reconstitution experiments, we show that SEC14-like undergoes liquid-like phase separation in the root stem cells. Outside the stem cell niche, SEC14-like associates with the caspase-like protease separase and conserved microtubule motors at unique polar plasma membrane interfaces. In these interfaces, SEC14-like undergoes abrupt processing by separase, which promotes its liquid-to-solid transition. The SEC14-like liquid-to-solid transition is important for root developmental robustness, as lines expressing an uncleavable SEC14-like variant or mutants of separase, and associated microtubule motors show similar developmental phenotypes. Furthermore, the processed and solidified but not the liquid form of SEC14-like interacts with the polar protein PINFORMED2 at the plasma membrane and perhaps other polar proteins of the PINFORMED family. This work demonstrates that robust development can involve abrupt liquid-to-solid transitions mediated by proteolysis at unique plasma membrane interfaces.

cell biology↗

bioRxiv2022

Arabidopsis PFA-DSP-type phosphohydrolases target specific inositol pyrophosphate messengers

Inositol pyrophosphates are signaling molecules containing at least one phosphoanhydride bond that regulate a wide range of cellular processes in eukaryotes. With a cyclic array of phosphate esters and diphosphate groups around myo-inositol, these molecular messengers possess the highest charge density found in nature. Recent work deciphering inositol pyrophosphate biosynthesis in Arabidopsis revealed important functions of these messengers in nutrient sensing, hormone signaling and plant immunity. However, despite the rapid hydrolysis of these molecules in plant extracts, very little is known about the molecular identity of the phosphohydrolases that convert these messengers back to their inositol polyphosphate precursors. Here, we investigate whether Arabidopsis Plant and Fungi Atypical Dual Specificity Phosphatases (PFA-DSP1-5) catalyze inositol pyrophosphate phosphohydrolase activity. We find that recombinant proteins of all five Arabidopsis PFA-DSP homologs display phosphohydrolase activity with a high specificity for the 5-{beta}-phosphate of inositol pyrophosphates. We further show that heterologous expression of Arabidopsis PFA-DSP1-5 rescues wortmannin-sensitivity and deranged inositol pyrophosphate homeostasis caused by the deficiency of the PFA-DSP-type inositol pyrophosphate phosphohydrolase Siw14 in yeast. Heterologous expression in Nicotiana benthamiana leaves provided evidence that Arabidopsis PFA-DSP1 also displays 5-{beta}-phosphate specific inositol pyrophosphate phosphohydrolase activity in planta. Our findings lay the biochemical basis and provide the genetic tools to uncover the roles of inositol pyrophosphates in plant physiology and plant development.

plant biology↗

bioRxiv2021

Arabidopsis inositol polyphosphate kinases IPK1 and ITPK1 modulate crosstalks between SA-dependent immunity and phosphate-starvation responses

The propensity for polyphosphorylation makes myo-inositol derivatives, the inositol polyphosphates (InsPs), especially phytic acid or inositol hexakisphosphate (InsP6) the major form of phosphate storage in plants. Acts of pyrophosphorylation on InsP6 generates InsP7 or InsP8 containing high-energy phosphoanhydride bonds that are harnessed during energy requirements of a cell. Also implicated as co-factors for several phytohormone signaling networks, InsP7/InsP8 modulate key developmental processes. With recent identification as the common moeity for transducing both jasmonic acid (JA) and phosphate-starvation responses (PSR), InsP8 is the classic example of a metabolite that may moonlight crosstalks to different cellular pathways during diverse stress adaptations. We show here that Arabidopsis thaliana INOSITOL PENTAKISPHOSPHATE 2-KINASE (IPK1), INOSITOL 1,3,4-TRISPHOSPHATE 5/6-KINASE 1 (ITPK1), and DIPHOSPHOINOSITOL PENTAKISPHOSPHATE KINASE 2 (VIH2), but not other InsP-kinases, suppress basal salicylic acid (SA)-dependent immunity. In ipk1, itpk1 or vih2 mutants, elevated endogenous SA levels and constitutive activation of defense signaling lead to enhanced resistance against the virulent Pseudomonas syringae pv tomato DC3000 (PstDC3000) strain. Our data reveal that activated SA-signaling sectors in these mutants modulate expression amplitudes of phosphate-starvation inducible (PSI)-genes, reported earlier. In turn, via mutualism the heightened basal defenses in these mutants require upregulated PSI-gene expressions likely highlighting the increased demand of phosphates required to support immunity. We demonstrate that SA is induced in phosphate-deprived plants, however its defense-promoting functions are likely diverted to PSR-supportive roles. Overall, our investigations reveal selective InsPs as crosstalk mediators among diverse signaling networks programming stress-appropriate adaptations.

plant biology↗

bioRxiv2020

Arabidopsis inositol polyphosphate kinases regulate COP9 signalosome functions in phosphate-homeostasis

Plant Cullin RING Ubiquitin E3 ligases (CRLs) facilitate targeted protein degradation during physiological development and adaptation to stress. The deneddylase activity of COP9 signalosome (CSN) regulates cellular ratios of neddylated cullins available for the continuum of CRL functions. While selective inositol polyphosphates (InsPs) function as co-factors in plant responses involving the ubiquitylation of negative regulators, a relationship to CSN-CRL activities has not yet been established. Here, we show that the two Arabidopsis thaliana InsP-kinases IPK1 and ITPK1 physically interact and metabolically connect with the CSN holo-complex to modulate cullin deneddylation efficiency. Specifically, functional deficiency of ITPK1 lowers cullin deneddylation rates and disrupts the dissociation equilibrium of CSN5, the deneddylase catalytic subunit, and CUL1 with the holo-complex. Our results identify a novel auto-regulatory switch of CSN functions, defined by deneddylation activity. We further demonstrate that phosphate starvation response (PSR), which is induced in Pi-starved wild-type plants and constitutive in the above InsP-kinase mutants, is orchestrated in part by reduced deneddylation rates that, in turn, affect the stability of SPX4, a key negative regulator of PSR. Pharmacological inhibition of cullin neddylation stabilizes SPX4 and impairs PSR, thus linking CSN-CRL dynamics to phosphate (Pi)-sensing. Conversely, when exposed to compounds that inhibit CSN5 deneddylase activity, wild-type plants display phenotypes similar to the above InsP-kinase mutants. Overall, our data reveal that the regulation of plant Pi-starvation responses by specific InsP-kinases is caused by a direct role of these kinases in balancing coordination between CRL-CSN activities.

plant biology↗

bioRxiv2020

Analysis of Inositol Phosphate Metabolism by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry (CE-ESI-MS)

The analysis of myo-inositol phosphates (InsPs) and myo-inositol pyrophosphates (PP-InsPs) is a daunting challenge due to the large number of possible isomers, the absence of a chromophore, the high charge density, the low abundance, and the instability of the esters and anhydrides. Given their importance in biology, an analytical approach to follow and understand this complex signaling hub is highly desirable. Here, capillary electrophoresis (CE) coupled to electrospray ionization mass spectrometry (ESI-MS) is implemented to analyze complex mixtures of InsPs and PP-InsPs with high sensitivity. Stable isotope labeled (SIL) internal standards allow for matrix-independent quantitative assignment. The method is validated in wild-type and knockout mammalian cell lines and in model organisms. SIL-CE-ESI-MS enables for the first time the accurate monitoring of InsPs and PP-InsPs arising from compartmentalized cellular synthesis pathways, by feeding cells with either [13C6]-myo-inositol or [13C6]-D-glucose. In doing so, we uncover that there must be unknown inositol synthesis pathways in mammals, highlighting the unique potential of this method to dissect inositol phosphate metabolism and signalling.

biochemistry↗

bioRxiv2020

ITPK1 is an InsP6/ADP phosphotransferase that controls systemic phosphate homeostasis in Arabidopsis

In plants, phosphate (Pi) homeostasis is regulated by the interaction of Pi starvation response transcription factors (PHRs) with stand-alone SPX proteins, which act as sensors for inositol pyrophosphates (PP-InsPs). Recently, ITPK1 was shown to generate the PP-InsP InsP7 from InsP6 in vitro, but the importance of this activity in Pi signaling remained unknown. Here, we show that uncontrolled Pi accumulation in ITPK1-deficient plants is accompanied by impaired Pi-dependent InsP7 and InsP8 synthesis. Reciprocal grafting demonstrates that Pi starvation responses are mainly controlled by ITPK1 activity in shoots. Nuclear magnetic resonance assays and PAGE analyses with recombinant protein reveal that besides InsP6 phosphorylation, ITPK1 is also able to catalyze ATP synthesis using 5-InsP7 but not any other InsP7 isomer as a P-donor when ATP is low. Additionally, we show that the dynamic changes in InsP7 and InsP8 to cellular Pi are conserved from land plant species to human cells, suggesting that Pi-dependent PP-InsP synthesis is a common component of Pi signaling across kingdoms. Together, our study demonstrates how Pi-dependent changes in nutritional and energetic states modulate ITPK1 activities to fine-tune the synthesis of PP-InsPs.

plant biology↗

bioRxiv2020

ITPK1-Dependent Inositol Polyphosphates Regulate Auxin Responses in Arabidopsis thaliana

The combinatorial phosphorylation of myo-inositol results in the generation of different inositol phosphates (InsP), of which phytic acid (InsP6) is the most abundant species in eukaryotes. InsP6 is also the precursor of higher phosphorylated forms called inositol pyrophosphates (PP-InsPs), such as InsP7 and InsP8, which are characterized by a diphosphate moiety and are also ubiquitously found in eukaryotic cells. While PP-InsPs regulate various cellular processes in animals and yeast, their biosynthesis and functions in plants has remained largely elusive because plant genomes do not encode canonical InsP6 kinases. Recently, it was shown that Arabidopsis ITPK1 catalyzes the phosphorylation of InsP6 to the natural 5-InsP7 isomer in vitro. Here, we demonstrate that Arabidopsis ITPK1 contributes to the synthesis of InsP7 in planta. We further find a critical role of ITPK1 in auxin-related processes including primary root elongation, leaf venation, thermomorphogenic and gravitropic responses, and sensitivity towards exogenously applied auxin. Notably, 5-InsP7 binds to recombinant auxin receptor complex, consisting of the F-Box protein TIR1, ASK1 and the transcriptional repressor IAA7, with high affinity. Furthermore, a specific increase in 5-InsP7 in a heterologous yeast expression system results in elevated interaction of the TIR1 homologs AFB1 and AFB2 with various AUX/IAA-type transcriptional repressors. We also identified a physical interaction between ITPK1 and TIR1, suggesting a dedicated channeling of an activating factor, such as 5-InsP7, to the auxin receptor complex. Our findings expand the mechanistic understanding of auxin perception and lay the biochemical and genetic basis to uncover physiological processes regulated by 5-InsP7.

plant biology↗

bioRxiv2020

Zebrafish macrophage developmental arrest underlies depletion of microglia and reveals Csf1r-independent metaphocytes

Macrophages derive from multiple sources of hematopoietic progenitors. Most macrophages require colony-stimulating factor 1 receptor (CSF1R), but some macrophages persist in the absence of CSF1R. Here, we analyzed mpeg1:GFP-expressing macrophages in csf1r-deficient zebrafish and report that embryonic macrophages emerge followed by their developmental arrest. In larvae, mpeg1+ cell numbers then increased showing two distinct types in the skin: branched, putative Langerhans cells, and amoeboid cells. In contrast, although numbers also increased in csf1r-mutants, exclusively amoeboid mpeg1+ cells were present, which we showed by genetic lineage tracing to have a non-hematopoietic origin. They expressed macrophage-associated genes, but also showed decreased phagocytic gene expression and increased epithelial-associated gene expression, characteristic of metaphocytes, recently discovered ectoderm-derived cells. We further demonstrated that juvenile csf1r-deficient zebrafish exhibit systemic macrophage depletion. Thus, Csf1r deficiency disrupts embryonic to adult macrophage development. Csf1r-deficient zebrafish are viable and permit analyzing the consequences of macrophage loss throughout life.

developmental biology↗