Publikation
Ruberti, C.; Feitosa-Araujo, E.; Xu, Z.; Wagner, S.; Grenzi, M.; Darwish, E.; Lichtenauer, S.; Fuchs, P.; Parmagnani, A. S.; Balcerowicz, D.; Schoenaers, S.; de la Torre, C.; Mekkaoui, K.; Nunes-Nesi, A.; Wirtz, M.; Vissenberg, K.; Van Aken, O.; Hause, B.; Costa, A.; Schwarzländer, M.; MCU proteins dominate in vivo mitochondrial Ca2+ uptake in Arabidopsis roots Plant Cell 34, 4428-4452, (2022) DOI: 10.1093/plcell/koac242
Abstract
Ca2+ signaling is central to plant development and acclimation. While Ca2+-responsive proteins have been investigated intensely in plants, only a few Ca2+-permeable channels have been identified, and our understanding of how intracellular Ca2+ fluxes is facilitated remains limited. Arabidopsis thaliana homologs of the mammalian channel-forming mitochondrial calcium uniporter (MCU) protein showed Ca2+ transport activity in vitro. Yet, the evolutionary complexity of MCU proteins, as well as reports about alternative systems and unperturbed mitochondrial Ca2+ uptake in knockout lines of MCU genes, leave critical questions about the in vivo functions of the MCU protein family in plants unanswered. Here, we demonstrate that MCU proteins mediate mitochondrial Ca2+ transport in planta and that this mechanism is the major route for fast Ca2+ uptake. Guided by the subcellular localization, expression, and conservation of MCU proteins, we generated an mcu triple knockout line. Using Ca2+ imaging in living root tips and the stimulation of Ca2+ transients of different amplitudes, we demonstrated that mitochondrial Ca2+ uptake became limiting in the triple mutant. The drastic cell physiological phenotype of impaired subcellular Ca2+ transport coincided with deregulated jasmonic acid-related signaling and thigmomorphogenesis. Our findings establish MCUs as a major mitochondrial Ca2+ entry route in planta and link mitochondrial Ca2+ transport with phytohormone signaling.
Publikation
Schubert, R.; Dobritzsch, S.; Gruber, C.; Hause, G.; Athmer, B.; Schreiber, T.; Marillonnet, S.; Okabe, Y.; Ezura, H.; Acosta, I. F.; Tarkowská, D.; Hause, B.; Tomato MYB21 Acts in Ovules to Mediate Jasmonate-Regulated Fertility Plant Cell 31, 1043-1062, (2019) DOI: 10.1105/tpc.18.00978
The function of the plant hormone jasmonic acid (JA) in the development of tomato (Solanum lycopersicum) flowers was analyzed with a mutant defective in JA perception (jasmonate-insensitive1-1, jai1-1). In contrast with Arabidopsis (Arabidopsis thaliana) JA-insensitive plants, which are male sterile, the tomato jai1-1 mutant is female sterile, with major defects in female development. To identify putative JA-dependent regulatory components, we performed transcriptomics on ovules from flowers at three developmental stages from wild type and jai1-1 mutants. One of the strongly downregulated genes in jai1-1 encodes the MYB transcription factor SlMYB21. Its Arabidopsis ortholog plays a crucial role in JA-regulated stamen development. SlMYB21 was shown here to exhibit transcription factor activity in yeast, to interact with SlJAZ9 in yeast and in planta, and to complement Arabidopsis myb21-5. To analyze SlMYB21 function, we generated clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR associated protein 9 (Cas9) mutants and identified a mutant by Targeting Induced Local Lesions in Genomes (TILLING). These mutants showed female sterility, corroborating a function of MYB21 in tomato ovule development. Transcriptomics analysis of wild type, jai1-1, and myb21-2 carpels revealed processes that might be controlled by SlMYB21. The data suggest positive regulation of JA biosynthesis by SlMYB21, but negative regulation of auxin and gibberellins. The results demonstrate that SlMYB21 mediates at least partially the action of JA and might control the flower-to-fruit transition.
Publikation
Matschi, S.; Hake, K.; Herde, M.; Hause, B.; Romeis, T.; The Calcium-Dependent Protein Kinase CPK28 Regulates Development by Inducing Growth Phase-Specific, Spatially Restricted Alterations in Jasmonic Acid Levels Independent of Defense Responses in Arabidopsis Plant Cell 27, 591-606, (2015) DOI: 10.1105/tpc.15.00024
Phytohormones play an important role in development and stress adaptations in plants, and several interacting hormonal pathways have been suggested to accomplish fine-tuning of stress responses at the expense of growth. This work describes the role played by the CALCIUM-DEPENDENT PROTEIN KINASE CPK28 in balancing phytohormone-mediated development in Arabidopsis thaliana, specifically during generative growth. cpk28 mutants exhibit growth reduction solely as adult plants, coinciding with altered balance of the phytohormones jasmonic acid (JA) and gibberellic acid (GA). JA-dependent gene expression and the levels of several JA metabolites were elevated in a growth phase-dependent manner in cpk28, and accumulation of JA metabolites was confined locally to the central rosette tissue. No elevated resistance toward herbivores or necrotrophic pathogens was detected for cpk28 plants, either on the whole-plant level or specifically within the tissue displaying elevated JA levels. Abolishment of JA biosynthesis or JA signaling led to a full reversion of the cpk28 growth phenotype, while modification of GA signaling did not. Our data identify CPK28 as a growth phase-dependent key negative regulator of distinct processes: While in seedlings, CPK28 regulates reactive oxygen species-mediated defense signaling; in adult plants, CPK28 confers developmental processes by the tissue-specific balance of JA and GA without affecting JA-mediated defense responses.
Publikation
Krajinski, F.; Courty, P.-E.; Sieh, D.; Franken, P.; Zhang, H.; Bucher, M.; Gerlach, N.; Kryvoruchko, I.; Zoeller, D.; Udvardi, M.; Hause, B.; The H+-ATPase HA1 of Medicago truncatula Is Essential for Phosphate Transport and Plant Growth during Arbuscular Mycorrhizal Symbiosis Plant Cell 26, 1808-1817, (2014) DOI: 10.1105/tpc.113.120436
A key feature of arbuscular mycorrhizal symbiosis is improved phosphorus nutrition of the host plant via the mycorrhizal pathway, i.e., the fungal uptake of Pi from the soil and its release from arbuscules within root cells. Efficient transport of Pi from the fungus to plant cells is thought to require a proton gradient across the periarbuscular membrane (PAM) that separates fungal arbuscules from the host cell cytoplasm. Previous studies showed that the H+-ATPase gene HA1 is expressed specifically in arbuscule-containing root cells of Medicago truncatula. We isolated a ha1-2 mutant of M. truncatula and found it to be impaired in the development of arbuscules but not in root colonization by Rhizophagus irregularis hyphae. Artificial microRNA silencing of HA1 recapitulated this phenotype, resulting in small and truncated arbuscules. Unlike the wild type, the ha1-2 mutant failed to show a positive growth response to mycorrhizal colonization under Pi-limiting conditions. Uptake experiments confirmed that ha1-2 mutants are unable to take up phosphate via the mycorrhizal pathway. Increased pH in the apoplast of abnormal arbuscule-containing cells of the ha1-2 mutant compared with the wild type suggests that HA1 is crucial for building a proton gradient across the PAM and therefore is indispensible for the transfer of Pi from the fungus to the plant.
Publikation
Ischebeck, T.; Werner, S.; Krishnamoorthy, P.; Lerche, J.; Meijon, M.; Stenzel, I.; Löfke, C.; Wiessner, T.; Im, Y. J.; Perera, I. Y.; Iven, T.; Feussner, I.; Busch, W.; Boss, W. F.; Teichmann, T.; Hause, B.; Persson, S.; Heilmann, I.; Phosphatidylinositol 4,5-Bisphosphate Influences PIN Polarization by Controlling Clathrin-Mediated Membrane Trafficking in Arabidopsis Plant Cell 25, 4894-4911, (2013) DOI: 10.1105/tpc.113.116582
The functions of the minor phospholipid phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] during vegetative plant growth remain obscure. Here, we targeted two related phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) PIP5K1 and PIP5K2, which are expressed ubiquitously in Arabidopsis thaliana. A pip5k1 pip5k2 double mutant with reduced PtdIns(4,5)P2 levels showed dwarf stature and phenotypes suggesting defects in auxin distribution. The roots of the pip5k1 pip5k2 double mutant had normal auxin levels but reduced auxin transport and altered distribution. Fluorescence-tagged auxin efflux carriers PIN-FORMED (PIN1)–green fluorescent protein (GFP) and PIN2-GFP displayed abnormal, partially apolar distribution. Furthermore, fewer brefeldin A–induced endosomal bodies decorated by PIN1-GFP or PIN2-GFP formed in pip5k1 pip5k2 mutants. Inducible overexpressor lines for PIP5K1 or PIP5K2 also exhibited phenotypes indicating misregulation of auxin-dependent processes, and immunolocalization showed reduced membrane association of PIN1 and PIN2. PIN cycling and polarization require clathrin-mediated endocytosis and labeled clathrin light chain also displayed altered localization patterns in the pip5k1 pip5k2 double mutant, consistent with a role for PtdIns(4,5)P2 in the regulation of clathrin-mediated endocytosis. Further biochemical tests on subcellular fractions enriched for clathrin-coated vesicles (CCVs) indicated that pip5k1 and pip5k2 mutants have reduced CCV-associated PI4P 5-kinase activity. Together, the data indicate an important role for PtdIns(4,5)P2 in the control of clathrin dynamics and in auxin distribution in Arabidopsis.
Publikation
Helber, N.; Wippel, K.; Sauer, N.; Schaarschmidt, S.; Hause, B.; Requena, N.; A Versatile Monosaccharide Transporter That Operates in the Arbuscular Mycorrhizal Fungus Glomus sp Is Crucial for the Symbiotic Relationship with Plants Plant Cell 23, 3812-3823, (2011) DOI: 10.1105/tpc.111.089813
For more than 400 million years, plants have maintained a mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi. This evolutionary success can be traced to the role of these fungi in providing plants with mineral nutrients, particularly phosphate. In return, photosynthates are given to the fungus, which support its obligate biotrophic lifestyle. Although the mechanisms involved in phosphate transfer have been extensively studied, less is known about the reciprocal transfer of carbon. Here, we present the high-affinity Monosaccharide Transporter2 (MST2) from Glomus sp with a broad substrate spectrum that functions at several symbiotic root locations. Plant cell wall sugars can efficiently outcompete the Glc uptake capacity of MST2, suggesting they can serve as alternative carbon sources. MST2 expression closely correlates with that of the mycorrhiza-specific PhosphateTransporter4 (PT4). Furthermore, reduction of MST2 expression using host-induced gene silencing resulted in impaired mycorrhiza formation, malformed arbuscules, and reduced PT4 expression. These findings highlight the symbiotic role of MST2 and support the hypothesis that the exchange of carbon for phosphate is tightly linked. Unexpectedly, we found that the external mycelium of AM fungi is able to take up sugars in a proton-dependent manner. These results imply that the sugar uptake system operating in this symbiosis is more complex than previously anticipated.
Publikation
Stenzel, I.; Ischebeck, T.; König, S.; Hołubowska, A.; Sporysz, M.; Hause, B.; Heilmann, I.; The Type B Phosphatidylinositol-4-Phosphate 5-Kinase 3 Is Essential for Root Hair Formation in Arabidopsis thaliana Plant Cell 20, 124-141, (2008) DOI: 10.1105/tpc.107.052852
Root hairs are extensions of root epidermal cells and a model system for directional tip growth of plant cells. A previously uncharacterized Arabidopsis thaliana phosphatidylinositol-4-phosphate 5-kinase gene (PIP5K3) was identified and found to be expressed in the root cortex, epidermal cells, and root hairs. Recombinant PIP5K3 protein was catalytically active and converted phosphatidylinositol-4-phosphate to phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2]. Arabidopsis mutant plants homozygous for T-DNA–disrupted PIP5K3 alleles were compromised in root hair formation, a phenotype complemented by expression of wild-type PIP5K3 cDNA under the control of a 1500-bp PIP5K3 promoter fragment. Root hair–specific PIP5K3 overexpression resulted in root hair deformation and loss of cell polarity with increasing accumulation of PIP5K3 transcript. Using reestablishment of root hair formation in T-DNA mutants as a bioassay for physiological functionality of engineered PIP5K3 variants, catalytic activity was found to be essential for physiological function, indicating that PtdIns(4,5)P2 formation is required for root hair development. An N-terminal domain containing membrane occupation and recognition nexus repeats, which is not required for catalytic activity, was found to be essential for the establishment of root hair growth. Fluorescence-tagged PIP5K3 localized to the periphery of the apical region of root hair cells, possibly associating with the plasma membrane and/or exocytotic vesicles. Transient heterologous expression of full-length PIP5K3 in tobacco (Nicotiana tabacum) pollen tubes increased plasma membrane association of a PtdIns(4,5)P2-specific reporter in these tip-growing cells. The data demonstrate that root hair development requires PIP5K3-dependent PtdIns(4,5)P2 production in the apical region of root hair cells.
Publikation
Binarová, P.; Cenklová, V.; Hause, B.; Kubátová, E.; Lysák, M.; Doležel, J.; Bögre, L.; Dráber, P.; Nuclear γ-Tubulin during Acentriolar Plant Mitosis Plant Cell 12, 433-442, (2000) DOI: 10.1105/tpc.12.3.433
Neither the molecular mechanism by which plant microtubules nucleate in the cytoplasm nor the organization of plant mitotic spindles, which lack centrosomes, is well understood. Here, using immunolocalization and cell fractionation techniques, we provide evidence that γ-tubulin, a universal component of microtubule organizing centers, is present in both the cytoplasm and the nucleus of plant cells. The amount of γ-tubulin in nuclei increased during the G2 phase, when cells are synchronized or sorted for particular phases of the cell cycle. γ-Tubulin appeared on prekinetochores before preprophase arrest caused by inhibition of the cyclin-dependent kinase and before prekinetochore labeling of the mitosis-specific phosphoepitope MPM2. The association of nuclear γ-tubulin with chromatin displayed moderately strong affinity, as shown by its release after DNase treatment and by using extraction experiments. Subcellular compartmentalization of γ-tubulin might be an important factor in the organization of plant-specific microtubule arrays and acentriolar mitotic spindles.