TY - JOUR ID - 2529 TI - Flexibility of intrinsically disordered degrons in AUX/IAA proteins reinforces auxin receptor assemblies JO - bioRxiv PY - 2019 SP - AU - Niemeyer, M. AU - Moreno Castillo, E. AU - Ihling, C. H. AU - Iacobucci, C. AU - Wilde, V. AU - Hellmuth, A. AU - Hoehenwarter, W. AU - Samodelov, S. L. AU - Zurbriggen, M. D. AU - Kastritis, P. L. AU - Sinz, A. AU - Calderón Villalobos, L. I. A. AU - VL - UR - DO - 10.1101/787770 AB - Cullin RING-type E3 ubiquitin ligases SCFTIR1/AFB1-5 and their ubiquitylation targets, AUX/IAAs, sense auxin concentrations in the nucleus. TIR1 binds a surface-exposed degron in AUX/IAAs promoting their ubiquitylation and rapid auxin-regulated proteasomal degradation. Here, we resolved 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. The AUX/IAA PB1 interaction domain also assists in non-native contacts, affecting AUX/IAA dynamic interaction states. Our results 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 adopting a multiplicity of functional states. We postulate IDRs in distinct members of the AUX/IAA family to be an adaptive signature for protein interaction and initiation region for proteasome recruitment. A2 - C1 - Molecular Signal Processing ER - TY - INPR ID - 2496 TI - Inositol pyrophosphates promote the interaction of SPX domains with the coiled-coil motif of PHR transcription factors to regulate plant phosphate homeostasis JO - bioRxiv PY - 2019 SP - AU - Ried, M. K. AU - Wild, R. AU - Zhu, J. AU - Broger, L. AU - Harmel, R. K. AU - Hothorn, L. A. AU - Fiedler, D. AU - Hothorn, M. AU - VL - UR - DO - 10.1101/2019.12.13.875393 AB - Phosphorus is an essential nutrient taken up by organisms in the form of inorganic phosphate (Pi). Eukaryotes have evolved sophisticated Pi sensing and signalling cascades, enabling them to maintain cellular Pi concentrations. Pi homeostasis is regulated by inositol pyrophosphate signalling molecules (PP-InsPs), which are sensed by SPX-domain containing proteins. In plants, PP-InsP bound SPX receptors inactivate Myb coiled-coil (MYB-CC) Pi starvation response transcription factors (PHRs) by an unknown mechanism. Here we report that a InsP8 – SPX complex targets the plant-unique CC domain of PHRs. Crystal structures of the CC domain reveal an unusual four-stranded anti-parallel arrangement. Interface mutations in the CC domain yield monomeric PHR1, which is no longer able to bind DNA with high affinity. Mutation of conserved basic residues located at the surface of the CC domain disrupt interaction with the SPX receptor in vitro and in planta, resulting in constitutive Pi starvation responses. Together, our findings suggest that InsP8 regulates plant Pi homeostasis by controlling the oligomeric state and hence the promoter binding capability of PHRs via their SPX receptors. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 750 TI - The missing link in jasmonic acid biosynthesis JO - Nat. Plants PY - 2019 SP - 776-777 AU - Wasternack, C. AU - Hause, B. AU - VL - 5 UR - DO - 10.1038/s41477-019-0492-y AB - Jasmonic acid biosynthesis starts in chloroplasts and is finalized in peroxisomes. The required export of a crucial intermediate out of the chloroplast is now shown to be mediated by a protein from the outer envelope called JASSY. A2 - C1 - Cell and Metabolic Biology; Molecular Signal Processing ER - TY - JOUR ID - 749 TI - Termination in Jasmonate Signaling by MYC2 and MTBs JO - Trends Plant Sci. PY - 2019 SP - 667-669 AU - Wasternack, C. AU - VL - 24 UR - DO - 10.1016/j.tplants.2019.06.001 AB - Jasmonic acid (JA) signaling can be switched off by metabolism of JA. The master regulator MYC2, interacting with MED25, has been shown to be deactivated by the bHLH transcription factors MTB1, MTB2, and MTB3. An autoregulatory negative feedback loop has been proposed for this termination in JA signaling. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 748 TI - New Light on Local and Systemic Wound Signaling JO - Trends Plant Sci. PY - 2019 SP - 102-105 AU - Wasternack, C. AU - VL - 24 UR - DO - 10.1016/j.tplants.2018.11.009 AB - Electric signaling and Ca2+ waves were discussed to occur in systemic wound responses. Two new overlapping scenarios were identified: (i) membrane depolarization in two special cell types followed by an increase in systemic cytoplasmic Ca2+ concentration ([Ca2+]cyt), and (ii) glutamate sensed by GLUTAMATE RECEPTOR LIKE proteins and followed by Ca2+-based defense in distal leaves. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 747 TI - Jasmonates are signals in the biosynthesis of secondary metabolites — Pathways, transcription factors and applied aspects — A brief review JO - New Biotechnol. PY - 2019 SP - 1-11 AU - Wasternack, C. AU - Strnad, M. AU - VL - 48 UR - DO - 10.1016/j.nbt.2017.09.007 AB - Jasmonates (JAs) are signals in plant stress responses and development. One of the first observed and prominent responses to JAs is the induction of biosynthesis of different groups of secondary compounds. Among them are nicotine, isoquinolines, glucosinolates, anthocyanins, benzophenanthridine alkaloids, artemisinin, and terpenoid indole alkaloids (TIAs), such as vinblastine. This brief review describes modes of action of JAs in the biosynthesis of anthocyanins, nicotine, TIAs, glucosinolates and artemisinin. After introducing JA biosynthesis, the central role of the SCFCOI1-JAZ co-receptor complex in JA perception and MYB-type and MYC-type transcription factors is described. Brief comments are provided on primary metabolites as precursors of secondary compounds. Pathways for the biosynthesis of anthocyanin, nicotine, TIAs, glucosinolates and artemisinin are described with an emphasis on JA-dependent transcription factors, which activate or repress the expression of essential genes encoding enzymes in the biosynthesis of these secondary compounds. Applied aspects are discussed using the biotechnological formation of artemisinin as an example of JA-induced biosynthesis of secondary compounds in plant cell factories. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 746 TI - Benno Parthier (1932–2019) JO - Plant Mol. Biol. PY - 2019 SP - 519-520 AU - Wasternack, C. AU - Hause, B. AU - Abel, S. AU - VL - 101 UR - DO - 10.1007/s11103-019-00927-6 AB - A2 - C1 - Cell and Metabolic Biology; Molecular Signal Processing ER - TY - JOUR ID - 732 TI - Wound-Induced Shoot-to-Root Relocation of JA-Ile Precursors Coordinates Arabidopsis Growth JO - Mol. Plant PY - 2019 SP - 1383-1394 AU - Schulze, A. AU - Zimmer, M. AU - Mielke, S. AU - Stellmach, H. AU - Melnyk, C. W. AU - Hause, B. AU - Gasperini, D. AU - VL - 12 UR - DO - 10.1016/j.molp.2019.05.013 AB - Multicellular organisms rely on the movement of signaling molecules across cells, tissues, and organs to communicate among distal sites. In plants, localized leaf damage activates jasmonic acid (JA)-dependent transcriptional reprogramming in both harmed and unharmed tissues. Although it has been indicated that JA species can translocate from damaged into distal sites, the identity of the mobile compound(s), the tissues through which they translocate, and the effect of their relocation remain unknown. Here, we found that following shoot wounding, the relocation of endogenous jasmonates through the phloem is essential to initiate JA signaling and stunt growth in unharmed roots of Arabidopsis thaliana. By employing grafting experiments and hormone profiling, we uncovered that the hormone precursor cis-12-oxo-phytodienoic acid (OPDA) and its derivatives, but not the bioactive JA-Ile conjugate, translocate from wounded shoots into undamaged roots. Upon root relocation, the mobile precursors cooperatively regulated JA responses through their conversion into JA-Ile and JA signaling activation. Collectively, our findings demonstrate the existence of long-distance translocation of endogenous OPDA and its derivatives, which serve as mobile molecules to coordinate shoot-to-root responses, and highlight the importance of a controlled redistribution of hormone precursors among organs during plant stress acclimation. A2 - C1 - Cell and Metabolic Biology; Molecular Signal Processing ER - TY - JOUR ID - 723 TI - Auxin-jasmonate crosstalk in Oryza sativa L. root system formation after cadmium and/or arsenic exposure JO - Environ. Exp. Bot. PY - 2019 SP - 59-69 AU - Ronzan, M. AU - Piacentini, D. AU - Fattorini, L. AU - Federica, D. R. AU - Caboni, E. AU - Eiche, E. AU - Ziegler, J. AU - Hause, B. AU - Riemann, M. AU - Betti, C. AU - Altamura, M. M. AU - Falasca, G. AU - VL - 165 UR - DO - 10.1016/j.envexpbot.2019.05.013 AB - Soil pollutants may affect root growth through interactions among phytohormones like auxin and jasmonates. Rice is frequently grown in paddy fields contaminated by cadmium and arsenic, but the effects of these pollutants on jasmonates/auxin crosstalk during adventitious and lateral roots formation are widely unknown. Therefore, seedlings of Oryza sativa cv. Nihonmasari and of the jasmonate-biosynthetic mutant coleoptile photomorphogenesis2 were exposed to cadmium and/or arsenic, and/or jasmonic acid methyl ester, and then analysed through morphological, histochemical, biochemical and molecular approaches.In both genotypes, arsenic and cadmium accumulated in roots more than shoots. In the roots, arsenic levels were more than twice higher than cadmium levels, either when arsenic was applied alone, or combined with cadmium. Pollutants reduced lateral root density in the wild -type in every treatment condition, but jasmonic acid methyl ester increased it when combined with each pollutant. Interestingly, exposure to cadmium and/or arsenic did not change lateral root density in the mutant. The transcript levels of OsASA2 and OsYUCCA2, auxin biosynthetic genes, increased in the wild-type and mutant roots when pollutants and jasmonic acid methyl ester were applied alone. Auxin (indole-3-acetic acid) levels transiently increased in the roots with cadmium and/or arsenic in the wild-type more than in the mutant. Arsenic and cadmium, when applied alone, induced fluctuations in bioactive jasmonate contents in wild-type roots, but not in the mutant. Auxin distribution was evaluated in roots of OsDR5::GUS seedlings exposed or not to jasmonic acid methyl ester added or not with cadmium and/or arsenic. The DR5::GUS signal in lateral roots was reduced by arsenic, cadmium, and jasmonic acid methyl ester. Lipid peroxidation, evaluated as malondialdehyde levels, was higher in the mutant than in the wild-type, and increased particularly in As presence, in both genotypes.Altogether, the results show that an auxin/jasmonate interaction affects rice root system development in the presence of cadmium and/or arsenic, even if exogenous jasmonic acid methyl ester only slightly mitigates pollutants toxicity. A2 - C1 - Cell and Metabolic Biology; Molecular Signal Processing ER - TY - JOUR ID - 721 TI - A set of Arabidopsis genes involved in the accommodation of the downy mildew pathogen Hyaloperonospora arabidopsidis JO - PLOS Pathog. PY - 2019 SP - e1007747 AU - Ried, M. K. AU - Banhara, A. AU - Hwu, F.-Y. AU - Binder, A. AU - Gust, A. A. AU - Höfle, C. AU - Hückelhoven, R. AU - Nürnberger, T. AU - Parniske, M. AU - VL - 15 UR - DO - 10.1371/journal.ppat.1007747 AB - The intracellular accommodation structures formed by plant cells to host arbuscular mycorrhiza fungi and biotrophic hyphal pathogens are cytologically similar. Therefore we investigated whether these interactions build on an overlapping genetic framework. In legumes, the malectin-like domain leucine-rich repeat receptor kinase SYMRK, the cation channel POLLUX and members of the nuclear pore NUP107-160 subcomplex are essential for symbiotic signal transduction and arbuscular mycorrhiza development. We identified members of these three groups in Arabidopsis thaliana and explored their impact on the interaction with the oomycete downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa). We report that mutations in the corresponding genes reduced the reproductive success of Hpa as determined by sporangiophore and spore counts. We discovered that a developmental transition of haustorial shape occurred significantly earlier and at higher frequency in the mutants. Analysis of the multiplication of extracellular bacterial pathogens, Hpa-induced cell death or callose accumulation, as well as Hpa- or flg22-induced defence marker gene expression, did not reveal any traces of constitutive or exacerbated defence responses. These findings point towards an overlap between the plant genetic toolboxes involved in the interaction with biotrophic intracellular hyphal symbionts and pathogens in terms of the gene families involved. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 669 TI - LCO Receptors Involved in Arbuscular Mycorrhiza Are Functional for Rhizobia Perception in Legumes JO - Curr. Biol. PY - 2019 SP - 4249-4259.e5 AU - Girardin, A. AU - Wang, T. AU - Ding, Y. AU - Keller, J. AU - Buendia, L. AU - Gaston, M. AU - Ribeyre, C. AU - Gasciolli, V. AU - Auriac, M.-C. AU - Vernié, T. AU - Bendahmane, A. AU - Ried, M. K. AU - Parniske, M. AU - Morel, P. AU - Vandenbussche, M. AU - Schorderet, M. AU - Reinhardt, D. AU - Delaux, P.-M. AU - Bono, J.-J. AU - Lefebvre, B. AU - VL - 29 UR - DO - 10.1016/j.cub.2019.11.038 AB - Bacterial lipo-chitooligosaccharides (LCOs) are key mediators of the nitrogen-fixing root nodule symbiosis (RNS) in legumes. The isolation of LCOs from arbuscular mycorrhizal fungi suggested that LCOs are also signaling molecules in arbuscular mycorrhiza (AM). However, the corresponding plant receptors have remained uncharacterized. Here we show that petunia and tomato mutants in the LysM receptor-like kinases LYK10 are impaired in AM formation. Petunia and tomato LYK10 proteins have a high affinity for LCOs (Kd in the nM range) comparable to that previously reported for a legume LCO receptor essential for the RNS. Interestingly, the tomato and petunia LYK10 promoters, when introduced into a legume, were active in nodules similarly to the promoter of the legume orthologous gene. Moreover, tomato and petunia LYK10 coding sequences restored nodulation in legumes mutated in their orthologs. This combination of genetic and biochemical data clearly pinpoints Solanaceous LYK10 as part of an ancestral LCO perception system involved in AM establishment, which has been directly recruited during evolution of the RNS in legumes. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 668 TI - Highly efficacious antiviral protection of plants by small interfering RNAs identified in vitro JO - Nucleic Acids Res. PY - 2019 SP - 9343-9357 AU - Gago-Zachert, S. AU - Schuck, J. AU - Weinholdt, C. AU - Knoblich, M. AU - Pantaleo, V. AU - Grosse, I. AU - Gursinsky, T. AU - Behrens, S.-E. AU - VL - 47 UR - DO - 10.1093/nar/gkz678 AB - In response to a viral infection, the plant’s RNA silencing machinery processes viral RNAs into a huge number of small interfering RNAs (siRNAs). However, a very few of these siRNAs actually interfere with viral replication. A reliable approach to identify these immunologically effective siRNAs (esiRNAs) and to define the characteristics underlying their activity has not been available so far. Here, we develop a novel screening approach that enables a rapid functional identification of antiviral esiRNAs. Tests on the efficacy of such identified esiRNAs of a model virus achieved a virtual full protection of plants against a massive subsequent infection in transient applications. We find that the functionality of esiRNAs depends crucially on two properties: the binding affinity to Argonaute proteins and the ability to access the target RNA. The ability to rapidly identify functional esiRNAs could be of great benefit for all RNA silencing-based plant protection measures against viruses and other pathogens. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 641 TI - Iron and Phosphate Deficiency Regulators Concertedly Control Coumarin Profiles in Arabidopsis thaliana Roots During Iron, Phosphate, and Combined Deficiencies JO - Front. Plant Sci. PY - 2019 SP - 113 AU - Chutia, R. AU - Abel, S. AU - Ziegler, J. AU - VL - 10 UR - DO - 10.3389/fpls.2019.00113 AB - Plants face varying nutrient conditions, to which they have to adapt to. Adaptive responses are nutrient-specific and strategies to ensure supply and homeostasis for one nutrient might be opposite to another one, as shown for phosphate (Pi) and iron (Fe) deficiency responses, where many genes are regulated in an opposing manner. This was also observed on the metabolite levels. Whereas root and exudate levels of catechol-type coumarins, phenylpropanoid-derived 2-benzopyranones, which facilitate Fe acquisition, are elevated after Fe deficiency, they are decreased after Pi deficiency. Exposing plants to combined Pi and Fe deficiency showed that the generation of coumarin profiles in Arabidopsis thaliana roots by Pi deficiency considerably depends on the availability of Fe. Similarly, the effect of Fe deficiency on coumarin profiles is different at low compared to high Pi availability. These findings suggest a fine-tuning of coumarin profiles, which depends on Fe and Pi availability. T-DNA insertion lines exhibiting aberrant expression of genes involved in the regulation of Pi starvation responses (PHO1, PHR1, bHLH32, PHL1, SPX1) and Fe starvation responses (BRUTUS, PYE, bHLH104, FIT) were used to analyze the regulation of the generation of coumarin profiles in Arabidopsis thaliana roots by Pi, Fe, and combined Pi and Fe deficiency. The analysis revealed a role of several Fe-deficiency response regulators in the regulation of Fe and of Pi deficiency-induced coumarin profiles as well as for Pi deficiency response regulators in the regulation of Pi and of Fe deficiency-induced coumarin profiles. Additionally, the regulation of Fe deficiency-induced coumarin profiles by Fe deficiency response regulators is influenced by Pi availability. Conversely, regulation of Pi deficiency-induced coumarin profiles by Pi deficiency response regulators is modified by Fe availability. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 703 TI - The Local Phosphate Deficiency Response Activates Endoplasmic Reticulum Stress-Dependent Autophagy JO - Plant Physiol. PY - 2019 SP - 460-476 AU - Naumann, C. AU - Müller, J. AU - Sakhonwasee, S. AU - Wieghaus, A. AU - Hause, G. AU - Heisters, M. AU - Bürstenbinder, K. AU - Abel, S. AU - VL - 179 UR - DO - 10.1104/pp.18.01379 AB - Inorganic phosphate (Pi) is often a limiting plant nutrient. In members of the Brassicaceae family, such as Arabidopsis (Arabidopsis thaliana), Pi deprivation reshapes root system architecture to favor topsoil foraging. It does so by inhibiting primary root extension and stimulating lateral root formation. Root growth inhibition from phosphate (Pi) deficiency is triggered by iron-stimulated, apoplastic reactive oxygen species generation and cell wall modifications, which impair cell-to-cell communication and meristem maintenance. These processes require LOW PHOSPHATE RESPONSE1 (LPR1), a cell wall-targeted ferroxidase, and PHOSPHATE DEFICIENCY RESPONSE2 (PDR2), the single endoplasmic reticulum (ER)-resident P5-type ATPase (AtP5A), which is thought to control LPR1 secretion or activity. Autophagy is a conserved process involving the vacuolar degradation of cellular components. While the function of autophagy is well established under nutrient starvation (C, N, or S), it remains to be explored under Pi deprivation. Because AtP5A/PDR2 likely functions in the ER stress response, we analyzed the effect of Pi limitation on autophagy. Our comparative study of mutants defective in the local Pi deficiency response, ER stress response, and autophagy demonstrated that ER stress-dependent autophagy is rapidly activated as part of the developmental root response to Pi limitation and requires the genetic PDR2-LPR1 module. We conclude that Pi-dependent activation of autophagy in the root apex is a consequence of local Pi sensing and the associated ER stress response, rather than a means for systemic recycling of the macronutrient. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 700 TI - Microtubule-associated protein IQ67 DOMAIN5 regulates morphogenesis of leaf pavement cells in Arabidopsis thaliana JO - J. Exp. Bot. PY - 2019 SP - 529-543 AU - Mitra, D. AU - Klemm, S. AU - Kumari, P. AU - Quegwer, J. AU - Möller, B. AU - Poeschl, Y. AU - Pflug, P. AU - Stamm, G. AU - Abel, S. AU - Bürstenbinder, K. AU - VL - 70 UR - DO - 10.1093/jxb/ery395 AB - Plant microtubules form a highly dynamic intracellular network with important roles for regulating cell division, cell proliferation and cell morphology. Its organization and dynamics are coordinated by various microtubule-associated proteins (MAPs) that integrate environmental and developmental stimuli to fine-tune and adjust cytoskeletal arrays. IQ67 DOMAIN (IQD) proteins recently emerged as a class of plant-specific MAPs with largely unknown functions. Here, using a reverse genetics approach, we characterize Arabidopsis IQD5 in terms of its expression domains, subcellular localization and biological functions. We show that IQD5 is expressed mostly in vegetative tissues, where it localizes to cortical microtubule arrays. Our phenotypic analysis of iqd5 loss-of-function lines reveals functions of IQD5 in pavement cell (PC) shape morphogenesis. Histochemical analysis of cell wall composition further suggests reduced rates of cellulose deposition in anticlinal cell walls, which correlate with reduced anisotropic expansion. Lastly, we demonstrate IQD5-dependent recruitment of calmodulin calcium sensors to cortical microtubule arrays and provide first evidence for important roles of calcium in regulation of PC morphogenesis. Our work thus identifies IQD5 as a novel player in PC shape regulation, and, for the first time, links calcium signaling to developmental processes that regulate anisotropic growth in PCs. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 699 TI - Interplay between Plant Cell Walls and Jasmonate Production JO - Plant Cell Physiol. PY - 2019 SP - 2629-2637 AU - Mielke, S. AU - Gasperini, D. AU - VL - 60 UR - DO - 10.1093/pcp/pcz119 AB - Plant cell walls are sophisticated carbohydrate-rich structures representing the immediate contact surface with the extracellular environment, often serving as the first barrier against biotic and abiotic stresses. Notably, a variety of perturbations in plant cell walls result in upregulated jasmonate (JA) production, a phytohormone with essential roles in defense and growth responses. Hence, cell wall-derived signals can initiate intracellular JA-mediated responses and the elucidation of the underlying signaling pathways could provide novel insights into cell wall maintenance and remodeling, as well as advance our understanding on how is JA biosynthesis initiated. This Mini Review will describe current knowledge about cell wall-derived damage signals and their effects on JA biosynthesis, as well as provide future perspectives. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 687 TI - Calcium- and calmodulin-regulated microtubule-associated proteins as signal-integration hubs at the plasma membrane–cytoskeleton nexus JO - J. Exp. Bot. PY - 2019 SP - 387-396 AU - Kölling, M. AU - Kumari, P. AU - Bürstenbinder, K. AU - VL - 70 UR - DO - 10.1093/jxb/ery397 AB - Plant growth and development are a genetically predetermined series of events but can change dramatically in response to environmental stimuli, involving perpetual pattern formation and reprogramming of development. The rate of growth is determined by cell division and subsequent cell expansion, which are restricted and controlled by the cell wall–plasma membrane–cytoskeleton continuum, and are coordinated by intricate networks that facilitate intra- and intercellular communication. An essential role in cellular signaling is played by calcium ions, which act as universal second messengers that transduce, integrate, and multiply incoming signals during numerous plant growth processes, in part by regulation of the microtubule cytoskeleton. In this review, we highlight recent advances in the understanding of calcium-mediated regulation of microtubule-associated proteins, their function at the microtubule cytoskeleton, and their potential role as hubs in crosstalk with other signaling pathways. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 685 TI - Studies of the Corneocytary Pathway Across the Stratum Corneum. Part I: Diffusion of Amino Acids Into the Isolated Corneocytes JO - Pharmazie PY - 2019 SP - 340-344 AU - Hussain, H. AU - Ziegler, J. AU - Mrestani, Y. AU - Neubert, R. H. H. AU - VL - 74 UR - DO - 10.1691/ph.2019.8098 AB - Amino acids (AAs), important constituents of natural moisturizing factors (NMFs) of the skin are decreased in diseased conditions such as psoriasis and atopic dermatitis. No study so far investigated the uptake of AAs into isolated corneocytes (COR). The present study was performed using 19 AAs, including taurine (TAU), to measure their amount diffused into the COR and binding of these AAs to keratin. Incubation of alanine, aspartic acid, asparagine, glutamine, glutamic acid, histidine, proline, serine and TAU with the isolated COR showed uptake after 24 h of 51.6, 95.4, 98.6, 94.1, 95.6, 90.1, 94.6, 72.9 and 57.8 %, respectively, into the COR but no binding with keratin. Uptake of TAU was validated by time dependent in-vitro diffusion models 'without COR and 'with COR'. The time dependent curve fitting showed that in in-vitro diffusion model 'without COR' there was no change in the total concentration of TAU until 72 hours, while in diffusion model 'with COR' the total conc. decreased to 37.8 % after 72 hours. The Pearson's correlation coefficient 'r' between the conc. curves of both in-vitro diffusion models was -0.54 that was an evidence of significant amount of TAU uptake by the COR. AAs as part of the NMFs have a great potential to be diffused into the COR. This property of the AAs can be employed in further dermatological research on diseased or aged skin conditions with NMFs deficiency. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 684 TI - Quantitative Analysis of Free Amino Acids and Urea Derived from Isolated Corneocytes of Healthy Young, Healthy Aged, and Diseased Skin JO - Skin Pharmacol. Physiol. PY - 2019 SP - 94-100 AU - Hussain, H. AU - Ziegler, J. AU - Hause, G. AU - Wohlrab, J. AU - Neubert, R. H. AU - VL - 32 UR - DO - 10.1159/000495992 AB - Background/Aims: Free amino acids (FAAs) and urea, present inside the corneocytes, can be important indicators of skin condition. However, due to the lack of a standard extraction protocol for FAAs from corneocytes, conflicting research results have been reported. Therefore, the purpose of this study was (1) to standardize the extraction protocol and (2) to investigate FAA profiles in healthy young and healthy old volunteers, as well as in psoriasis and atopic dermatitis patients. Methods: Skin samples were collected from four groups (healthy young, healthy old, and psoriasis and atopic dermatitis patients) with 5 volunteers per group. Corneocytes were isolated and examined microscopically. FAAs and urea were extracted from the isolated corneocytes, and their amounts were quantified using LC-ESI/MS/MS (after derivatization with Fmoc-Cl) and colorimetric methods, respectively. Results: The micrographs of the corneocytes showed no morphological features attributable to age or disease conditions. The highest and lowest concentrations of total FAAs and urea were observed in the healthy old group and the healthy young group, respectively. Unlike the other FAAs and urea, citrulline was found at a higher level in the healthy young group than in the disease groups. Conclusion: This study suggests that the levels of FAAs and urea in the skin are affected by age and skin conditions (healthy/diseased). However, further studies are needed to show the effects of different skin conditions on the levels of FAAs and urea. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 636 TI - Detection of MCPG metabolites in horses with atypical myopathy JO - PLOS ONE PY - 2019 SP - e0211698 AU - Bochnia, M. AU - Sander, J. AU - Ziegler, J. AU - Terhardt, M. AU - Sander, S. AU - Janzen, N. AU - Cavalleri, J.-M. V. AU - Zuraw, A. AU - Wensch-Dorendorf, M. AU - Zeyner, A. AU - VL - 14 UR - DO - 10.1371/journal.pone.0211698 AB - Atypical myopathy (AM) in horses is caused by ingestion of seeds of the Acer species (Sapindaceae family). Methylenecyclopropylacetyl-CoA (MCPA-CoA), derived from hypoglycin A (HGA), is currently the only active toxin in Acer pseudoplatanus or Acer negundo seeds related to AM outbreaks. However, seeds or arils of various Sapindaceae (e.g., ackee, lychee, mamoncillo, longan fruit) also contain methylenecyclopropylglycine (MCPG), which is a structural analogue of HGA that can cause hypoglycaemic encephalopathy in humans. The active poison formed from MCPG is methylenecyclopropylformyl-CoA (MCPF-CoA). MCPF-CoA and MCPA-CoA strongly inhibit enzymes that participate in β-oxidation and energy production from fat. The aim of our study was to investigate if MCPG is involved in Acer seed poisoning in horses. MCPG, as well as glycine and carnitine conjugates (MCPF-glycine, MCPF-carnitine), were quantified using high-performance liquid chromatography-tandem mass spectrometry of serum and urine from horses that had ingested Acer pseudoplatanus seeds and developed typical AM symptoms. The results were compared to those of healthy control horses. For comparison, HGA and its glycine and carnitine derivatives were also measured. Additionally, to assess the degree of enzyme inhibition of β-oxidation, several acyl glycines and acyl carnitines were included in the analysis. In addition to HGA and the specific toxic metabolites (MCPA-carnitine and MCPA-glycine), MCPG, MCPF-glycine and MCPF-carnitine were detected in the serum and urine of affected horses. Strong inhibition of β-oxidation was demonstrated by elevated concentrations of all acyl glycines and carnitines, but the highest correlations were observed between MCPF-carnitine and isobutyryl-carnitine (r = 0.93) as well as between MCPA- (and MCPF-) glycine and valeryl-glycine with r = 0.96 (and r = 0.87). As shown here, for biochemical analysis of atypical myopathy of horses, it is necessary to take MCPG and the corresponding metabolites into consideration. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 634 TI - Balancing trade-offs between biotic and abiotic stress responses through leaf age-dependent variation in stress hormone cross-talk JO - Proc. Natl. Acad. Sci. U.S.A. PY - 2019 SP - 2364-2373 AU - Berens, M. L. AU - Wolinska, K. W. AU - Spaepen, S. AU - Ziegler, J. AU - Nobori, T. AU - Nair, A. AU - Krüler, V. AU - Winkelmüller, T. M. AU - Wang, Y. AU - Mine, A. AU - Becker, D. AU - Garrido-Oter, R. AU - Schulze-Lefert, P. AU - Tsuda, K. AU - VL - 116 UR - DO - 10.1073/pnas.1817233116 AB - In nature, plants must respond to multiple stresses simultaneously, which likely demands cross-talk between stress-response pathways to minimize fitness costs. Here we provide genetic evidence that biotic and abiotic stress responses are differentially prioritized in Arabidopsis thaliana leaves of different ages to maintain growth and reproduction under combined biotic and abiotic stresses. Abiotic stresses, such as high salinity and drought, blunted immune responses in older rosette leaves through the phytohormone abscisic acid signaling, whereas this antagonistic effect was blocked in younger rosette leaves by PBS3, a signaling component of the defense phytohormone salicylic acid. Plants lacking PBS3 exhibited enhanced abiotic stress tolerance at the cost of decreased fitness under combined biotic and abiotic stresses. Together with this role, PBS3 is also indispensable for the establishment of salt stress- and leaf age-dependent phyllosphere bacterial communities. Collectively, our work reveals a mechanism that balances trade-offs upon conflicting stresses at the organism level and identifies a genetic intersection among plant immunity, leaf microbiota, and abiotic stress tolerance. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 633 TI - A Mobile Auxin Signal Connects Temperature Sensing in Cotyledons with Growth Responses in Hypocotyls JO - Plant Physiol. PY - 2019 SP - 757-766 AU - Bellstaedt, J. AU - Trenner, J. AU - Lippmann, R. AU - Poeschl, Y. AU - Zhang, X. AU - Friml, J. AU - Quint, M. AU - Delker, C. AU - VL - 180 UR - DO - 10.1104/pp.18.01377 AB - Plants have a remarkable capacity to adjust their growth and development to elevated ambient temperatures. Increased elongation growth of roots, hypocotyls, and petioles in warm temperatures are hallmarks of seedling thermomorphogenesis. In the last decade, significant progress has been made to identify the molecular signaling components regulating these growth responses. Increased ambient temperature utilizes diverse components of the light sensing and signal transduction network to trigger growth adjustments. However, it remains unknown whether temperature sensing and responses are universal processes that occur uniformly in all plant organs. Alternatively, temperature sensing may be confined to specific tissues or organs, which would require a systemic signal that mediates responses in distal parts of the plant. Here, we show that Arabidopsis (Arabidopsis thaliana) seedlings show organ-specific transcriptome responses to elevated temperatures and that thermomorphogenesis involves both autonomous and organ-interdependent temperature sensing and signaling. Seedling roots can sense and respond to temperature in a shoot-independent manner, whereas shoot temperature responses require both local and systemic processes. The induction of cell elongation in hypocotyls requires temperature sensing in cotyledons, followed by the generation of a mobile auxin signal. Subsequently, auxin travels to the hypocotyl, where it triggers local brassinosteroid-induced cell elongation in seedling stems, which depends upon a distinct, permissive temperature sensor in the hypocotyl. A2 - C1 - Molecular Signal Processing ER - TY - CHAP ID - 34 TI - Sensitive and Selective Amino Acid Profiling of Minute Tissue Amounts by HPLC/Electrospray Negative Tandem Mass Spectrometry Using 9-Fluorenylmethoxycarbonyl (Fmoc-Cl) Derivatization T2 - Amino Acid Analysis PB - Methods Mol. Biol. PY - 2019 SP - 365-379 AU - Ziegler, J. AU - Hussain, H. AU - Neubert, R. H. H. AU - Abel, S. AU - VL - 2030 UR - SN - 978-1-4939-9639-1 DO - 10.1007/978-1-4939-9639-1_27 AB - A method for selective and sensitive quantification of amino acids is described. The combination of established derivatization procedures of secondary and primary amino groups with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) and subsequent detection of derivatized amino acids by LC-ESI-MS/MS using multiple reaction monitoring provides high selectivity. The attachment of an apolar moiety enables purification of derivatized amino acids from matrix by a single solid-phase extraction step, which increases sensitivity by reduced ion suppression during LC-ESI-MS/MS detection. Additionally, chromatography of all amino acids can be performed on reversed-phase HPLC columns using eluents without additives, which are known to cause significant decreases in signal to noise ratios. The method has been routinely applied for amino acid profiling of low amounts of liquids and tissues of various origins with a sample throughput of about 50–100 samples a day. In addition to a detailed description of the method, some representative examples are presented. A2 - Alterman, M. A., ed. C1 - Molecular Signal Processing ER - TY - CHAP ID - 31 TI - Semi-Automatic Cell Segmentation from Noisy Image Data for Quantification of Microtubule Organization on Single Cell Level T2 - 2019 IEEE 16th International Symposium on Biomedical Imaging (ISBI 2019) PB - PY - 2019 SP - 199-203 AU - Möller, B. AU - Bürstenbinder, K. AU - VL - UR - SN - 978-1-5386-3641-1 DO - 10.1109/ISBI.2019.8759145 AB - The structure of the microtubule cytoskeleton provides valuable information related to morphogenesis of cells. The cytoskeleton organizes into diverse patterns that vary in cells of different types and tissues, but also within a single tissue. To assess differences in cytoskeleton organization methods are needed that quantify cytoskeleton patterns within a complete cell and which are suitable for large data sets. A major bottleneck in most approaches, however, is a lack of techniques for automatic extraction of cell contours. Here, we present a semi-automatic pipeline for cell segmentation and quantification of microtubule organization. Automatic methods are applied to extract major parts of the contours and a handy image editor is provided to manually add missing information efficiently. Experimental results prove that our approach yields high-quality contour data with minimal user intervention and serves a suitable basis for subsequent quantitative studies. A2 - C1 - Molecular Signal Processing ER - TY - CHAP ID - 30 TI - Quantitative and Comparative Analysis of Global Patterns of (Microtubule) Cytoskeleton Organization with CytoskeletonAnalyzer2D T2 - Plant Cell Morphogenesis PB - Methods Mol. Biol. PY - 2019 SP - 151-171 AU - Möller, B. AU - Zergiebel, L. AU - Bürstenbinder, K. AU - VL - 1992 UR - SN - 978-1-4939-9469-4 DO - 10.1007/978-1-4939-9469-4_10 AB - The microtubule cytoskeleton plays important roles in cell morphogenesis. To investigate the mechanisms of cytoskeletal organization, for example, during growth or development, in genetic studies, or in response to environmental stimuli, image analysis tools for quantitative assessment are needed. Here, we present a method for texture measure-based quantification and comparative analysis of global microtubule cytoskeleton patterns and subsequent visualization of output data. In contrast to other approaches that focus on the extraction of individual cytoskeletal fibers and analysis of their orientation relative to the growth axis, CytoskeletonAnalyzer2D quantifies cytoskeletal organization based on the analysis of local binary patterns. CytoskeletonAnalyzer2D thus is particularly well suited to study cytoskeletal organization in cells where individual fibers are difficult to extract or which lack a clearly defined growth axis, such as leaf epidermal pavement cells. The tool is available as ImageJ plugin and can be combined with publicly available software and tools, such as R and Cytoscape, to visualize similarity networks of cytoskeletal patterns. A2 - Cvrčková, F. & Žárský, V., eds. C1 - Molecular Signal Processing ER - TY - CHAP ID - 29 TI - Morphological Analysis of Leaf Epidermis Pavement Cells with PaCeQuant T2 - Plant Cell Morphogenesis PB - Methods Mol. Biol. PY - 2019 SP - 329-349 AU - Möller, B. AU - Poeschl, Y. AU - Klemm, S. AU - Bürstenbinder, K. AU - VL - 1992 UR - SN - 978-1-4939-9469-4 DO - 10.1007/978-1-4939-9469-4_22 AB - Morphological analysis of cell shapes requires segmentation of cell contours from input images and subsequent extraction of meaningful shape descriptors that provide the basis for qualitative and quantitative assessment of shape characteristics. Here, we describe the publicly available ImageJ plugin PaCeQuant and its associated R package PaCeQuantAna, which provides a pipeline for fully automatic segmentation, feature extraction, statistical analysis, and graphical visualization of cell shape properties. PaCeQuant is specifically well suited for analysis of jigsaw puzzle-like leaf epidermis pavement cells from 2D input images and supports the quantification of global, contour-based, skeleton-based, and pavement cell-specific shape descriptors. A2 - Cvrčková, F. & Žárský, V., eds. C1 - Molecular Signal Processing ER - TY - JOUR ID - 2293 TI - Molecular Cloning of Allene Oxide Cyclase JO - J. Biol. Chem. PY - 2000 SP - 19132-19138 AU - Ziegler, J. AU - Stenzel, I. AU - Hause, B. AU - Maucher, H. AU - Hamberg, M. AU - Grimm, R. AU - Ganal, M. AU - Wasternack, C. AU - VL - 275 UR - DO - 10.1074/jbc.M002133200 AB - Allene oxide cyclase (EC 5.3.99.6) catalyzes the stereospecific cyclization of an unstable allene oxide to (9S,13S)-12-oxo-(10,15Z)-phytodienoic acid, the ultimate precursor of jasmonic acid. This dimeric enzyme has previously been purified, and two almost identical N-terminal peptides were found, suggesting allene oxide cyclase to be a homodimeric protein. Furthermore, the native protein was N-terminally processed. Using degenerate primers, a polymerase chain reaction fragment could be generated from tomato, which was further used to isolate a full-length cDNA clone of 1 kilobase pair coding for a protein of 245 amino acids with a molecular mass of 26 kDa. Whereas expression of the whole coding region failed to detect allene oxide cyclase activity, a 5′-truncated protein showed high activity, suggesting that additional amino acids impair the enzymatic function. Steric analysis of the 12-oxophytodienoic acid formed by the recombinant enzyme revealed exclusive (>99%) formation of the 9S,13Senantiomer. Exclusive formation of this enantiomer was also found in wounded tomato leaves. Southern analysis and genetic mapping revealed the existence of a single gene for allene oxide cyclase located on chromosome 2 of tomato. Inspection of the N terminus revealed the presence of a chloroplastic transit peptide, and the location of allene oxide cyclase protein in that compartment could be shown by immunohistochemical methods. Concomitant with the jasmonate levels, the accumulation of allene oxide cyclase mRNA was transiently induced after wounding of tomato leaves. A2 - C1 - Molecular Signal Processing; Cell and Metabolic Biology ER - TY - JOUR ID - 2290 TI - Formation of 4-hydroxy-2-alkenals in barley leaves JO - Biochem. Soc. Trans. PY - 2000 SP - 850-851 AU - Weichert, H. AU - Kolbe, A. AU - Wasternack, C. AU - Feussner, I. AU - VL - 28 UR - DO - 10.1042/bst0280850 AB - In barley leaves 13-lipoxygenases are induced by jasmonates. This leads to induction of lipid peroxidation. Here we show by in vitro studies that these processes may further lead to autoxidative formation of (2E)-4-hydroxy-2-hexenal from (3Z)-hexenal. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2289 TI - Stressabwehr und Entwicklung: Jasmonate — chemische Signale in Pflanzen JO - Biologie in unserer Zeit PY - 2000 SP - 312-320 AU - Wasternack, C. AU - Hause, B. AU - VL - 30 UR - DO - 10.1002/1521-415X(200011)30:6<312::AID-BIUZ312>3.0.CO;2-8 AB - Chemische Signale wurden bereits im 19.Jahrhundert als Regulatoren von Wachstum und Entwicklung der Pflanzen postuliert.In den letzten 70 Jahren wurde die Wirkungsweise der klassischen Pflanzenhormone wie der Auxine, Gibberelline, Cytokinine, Ethylen und Abscisinsäure aufgeklärt. Doch erst im letzten Jahrzehnt entdeckte man die Bedeutung der Brassinosteroide, der Peptidhormone und der Jasmonate. A2 - C1 - Molecular Signal Processing; Cell and Metabolic Biology ER - TY - JOUR ID - 2279 TI - Breeding for virus resistance in maize JO - Genetika PY - 2000 SP - 529-545 AU - Quint, M. AU - Melchinger, A. E. AU - Dußle, C. M. AU - Lübberstedt, T. AU - VL - 32 UR - http://www.dgsgenetika.org.rs/abstrakti/vol32_2000_no3_en.htm#Rad26 AB - Sugarcane mosaic virus (SCMV) is an important disease in maize, which is emerging in Germany since 1983. Using this pest as a model for the inheritance of oligogenic traits, we clarified the genetic ba­sis for resistance in early maturing European maize germplasm. Screening of 122 adapted European inbred lines identified three completely resistant lines, which were used for further analyses. The genetics of SCMV resis­tance was investigated by allelism tests in field experiments combined with QTL and bulked segregant analyses (BSA) on the marker level. QTL analyses revealed the presence of two major genes Scm1 and Scm2 plus three minor QTL. Involvement of Scm1 and Scm2 in the inheritance of SCMV resistance could be confirmed by BSA in a second cross. Breeders can make use of tightly linked STS markers for marker-assisted selection (MAS) as well as our SCMV resistant flint lines to improve their elite germplasm. Currently, recurrent backcrossing with phenotypic selection is the most appropriate and cost effective breeding method. With de­creasing costs of DNA chip technology, MAS can be competitive with phenotypic selection in the near future. Further objectives of our research are the isolation and cloning of Scm1 and Scm2. To achieve this goal we follow two different approaches. (1) Positional cloning based on more than 500 AFLP primer combinations resulted in Scm1/Scm2 specific markers with a resolution of approximately 0.2 cM in the respective re­gions. (2) Resistance gene analogues (RGAs), cosegregating with the tar­get genes are used to identify further candidate genes for transformation experiments. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2272 TI - Octadecanoid and Jasmonate Signaling in Tomato (Lycopersicon esculentum Mill.) Leaves: Endogenous Jasmonates Do Not Induce Jasmonate Biosynthesis JO - Biol. Chem. PY - 2000 SP - 715-722 AU - Miersch, O. AU - Wasternack, C. AU - VL - 381 UR - DO - 10.1515/BC.2000.092 AB - Jasmonates and their precursors, the octadecanoids, are signals in stress-induced alteration of gene expression. Several mRNAs coding for enzymes of jasmonic acid (JA) biosynthesis are up-regulated upon JA treatment or endogenous increase of the JA level. Here we investigated the positive feedback of endogenous JA on JA formation, as well as its β-oxidation steps. JA-responsive gene expression was recorded in terms of proteinase inhibitor2 (pin2) mRNA accumulation. JA formed upon treatment of tomato (Lycopersicon esculentum cv. Moneymaker) leaves with JA derivatives carrying different lengths of the carboxylic acid side chain was quantified by gas chromatography-mass spectrometry (GC-MS). The data revealed that β-oxidation of the side chain occurs up to a butyric acid moiety. The amount of JA formed from side-chain modified JA derivatives correlated with pin2-mRNA accumulation. JA derivatives with a carboxylic side chain of 3, 5 or 7 carbon atoms were unable to form JA and to express on pin2, whereas evennumbered derivatives were active.After treatment of tomato leaves with (10-2H)-(–)-12-oxophytoenoic acid, (4-2H)-(–)-JA and its methyl ester were formed and could be quantified separately from the endogenously nonlabeled JA pool by GC-MS analysis via isotopic discrimination. The level of 8 nmol per g fresh weight JA and its methyl ester originated exclusively from labeled 12-oxophytoenic acid. This and further data indicate that endogenous synthesis of the JA precursor 12-oxophytodienoic acid, as well as of JA and its methyl ester, are not induced in tomato leaves, suggesting that positive feedback in JA biosynthesis does not function in vivo. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2271 TI - Allene oxide synthases of barley (Hordeum vulgare cv. Salome): tissue specific regulation in seedling development JO - Plant J. PY - 2000 SP - 199-213 AU - Maucher, H. AU - Hause, B. AU - Feussner, I. AU - Ziegler, J. AU - Wasternack, C. AU - VL - 21 UR - DO - 10.1046/j.1365-313x.2000.00669.x AB - Allene oxide synthase (AOS) is the first enzyme in the lipoxygenase (LOX) pathway which leads to formation of jasmonic acid (JA). Two full‐length cDNAs of AOS designated as AOS1 and AOS2, respectively, were isolated from barley (H. vulgare cv. Salome) leaves, which represent the first AOS clones from a monocotyledonous species. For AOS1, the open reading frame encompasses 1461 bp encoding a polypeptide of 487 amino acids with calculated molecular mass of 53.4 kDa and an isoelectric point of 9.3, whereas the corresponding data of AOS2 are 1443 bp, 480 amino acids, 52.7 kDa and 7.9. Southern blot analysis revealed at least two genes. Despite the lack of a putative chloroplast signal peptide in both sequences, the protein co‐purified with chloroplasts and was localized within chloroplasts by immunocytochemical analysis. The barley AOSs, expressed in bacteria as active enzymes, catalyze the dehydration of LOX‐derived 9‐ as well as 13‐hydroperoxides of polyenoic fatty acids to the unstable allene oxides. In leaves, AOS mRNA accumulated upon treatment with jasmonates, octadecanoids and metabolizable carbohydrates, but not upon floating on abscisic acid, NaCl, Na‐salicylate or infection with powdery mildew. In developing seedlings, AOS mRNA strongly accumulated in the scutellar nodule, but less in the leaf base. Both tissues exhibited elevated JA levels. In situ hybridizations revealed the preferential occurrence of AOS mRNA in parenchymatic cells surrounding the vascular bundles of the scutellar nodule and in the young convoluted leaves as well as within the first internode. The properties of both barley AOSs, their up‐regulation of their mRNAs and their tissue specific expression suggest a role during seedling development and jasmonate biosynthesis. A2 - C1 - Molecular Signal Processing; Cell and Metabolic Biology ER - TY - JOUR ID - 2266 TI - Octadecanoid-Derived Alteration of Gene Expression and the “Oxylipin Signature” in Stressed Barley Leaves. Implications for Different Signaling Pathways JO - Plant Physiol. PY - 2000 SP - 177-188 AU - Kramell, R. AU - Miersch, O. AU - Atzorn, R. AU - Parthier, B. AU - Wasternack, C. AU - VL - 123 UR - DO - 10.1104/pp.123.1.177 AB - Stress-induced gene expression in barley (Hordeum vulgare cv Salome) leaves has been correlated with temporally changing levels of octadecanoids and jasmonates, quantified by means of gas chromatography/mass spectrometry-single ion monitoring. Application of sorbitol-induced stress led to a low and transient rise of jasmonic acid (JA), its precursor 12-oxophytodienoic acid (OPDA), and the methyl esters JAME and OPDAME, respectively, followed by a large increase in their levels. JA and JAME peaked between 12 and 16 h, about 4 h before OPDA and OPDAME. However, OPDA accumulated up to a 2.5-fold higher level than the other compounds. Dihomo-JA and 9,13-didehydro-OPDA were identified as minor components. Kinetic analyses revealed that a transient threshold of jasmonates or octadecanoids is necessary and sufficient to initiate JA-responsive gene expression. Although OPDA and OPDAME applied exogenously were metabolized to JA in considerable amounts, both of them can induce gene expression, as evidenced by those genes that did not respond to endogenously formed JA. Also, coronatine induces JA-responsive genes independently from endogenous JA. Application of deuterated JA showed that endogenous synthesis of JA is not induced by JA treatment. The data are discussed in terms of distinct signaling pathways. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2258 TI - Tissue-specific oxylipin signature of tomato flowers: allene oxide cyclase is highly expressed in distinct flower organs and vascular bundles JO - Plant J. PY - 2000 SP - 113-126 AU - Hause, B. AU - Stenzel, I. AU - Miersch, O. AU - Maucher, H. AU - Kramell, R. AU - Ziegler, J. AU - Wasternack, C. AU - VL - 24 UR - DO - 10.1046/j.1365-313x.2000.00861.x AB - A crucial step in the biosynthesis of jasmonic acid (JA) is the formation of its correct stereoisomeric precursor, cis (+)12‐oxophytodienoic acid (OPDA). This step is catalysed by allene oxide cyclase (AOC), which has been recently cloned from tomato . In stems, young leaves and young flowers, AOC mRNA accumulates to a low level , contrasting with a high accumulation in flower buds, flower stalks and roots. The high levels of AOC mRNA and AOC protein in distinct flower organs correlate with high AOC activity, and with elevated levels of JA, OPDA and JA isoleucine conjugate. These compounds accumulate in flowers to levels of about 20 nmol g−1 fresh weight, which is two orders of magnitude higher than in leaves. In pistils, the level of OPDA is much higher than that of JA, whereas in flower stalks, the level of JA exceeds that of OPDA. In other flower tissues, the ratios among JA, OPDA and JA isoleucine conjugate differ remarkably, suggesting a tissue‐specific oxylipin signature. Immunocytochemical analysis revealed the specific occurrence of the AOC protein in ovules, the transmission tissue of the style and in vascular bundles of receptacles, flower stalks, stems, petioles and roots. Based on the tissue‐specific AOC expression and formation of JA, OPDA and JA amino acid conjugates, a possible role for these compounds in flower development is discussed in terms of their effect on sink–source relationships and plant defence reactions. Furthermore, the AOC expression in vascular bundles might play a role in the systemin‐mediated wound response of tomato. A2 - C1 - Molecular Signal Processing; Cell and Metabolic Biology ER - TY - JOUR ID - 2256 TI - Functional detection of chemopreventive glucosinolates in Arabidopsis thaliana JO - Plant Sci. PY - 2000 SP - 265-272 AU - Gross, H. B. AU - Dalebout, T. AU - Grubb, C. D. AU - Abel, S. AU - VL - 159 UR - DO - 10.1016/S0168-9452(00)00354-X AB - Natural isothiocyanates, derived from glucosinolates by myrosinase-catalyzed hydrolysis, are potent chemopreventive agents that favorably modify carcinogen metabolism in mammals by inhibiting metabolic activation of carcinogens and/or by inducing carcinogen-detoxifying enzymes. Methylsulfinylalkyl isothiocyanates are potent selective inducers of mammalian Phase 2 detoxification enzymes such as quinone reductase [NADP(H):quinone-acceptor oxidoreductase, EC 1.6.99.2]. Members of the Cruciferae family, including the model plant species Arabidopsis thaliana (L.) Heyhn, synthesize methylsulfinylalkyl glucosinolates. We have adapted a colorimetric bioassay for quinone reductase activity in Hepa 1c1c7 murine hepatoma cells as a versatile tool to rapidly monitor methylsulfinylalkyl glucosinolate content in A. thaliana leaf extracts. Using wild type plants and mutant plants defective in the synthesis of 4-methylsulfinylbutyl glucosinolate (glucoraphanin), we have demonstrated that A. thaliana (ecotype Columbia) is a rich source of Phase 2 enzyme inducers and that methylsulfinylalkyl glucosinolates, predominantly glucoraphanin, account for about 80% of the quinone reductase inducer potency of Columbia leaf extracts. We have optimized leaf extraction conditions and the quinone reductase bioassay to allow for screening of large numbers of plant extracts in a molecular genetic approach to dissecting glucosinolate biosynthesis in A. thaliana. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2254 TI - Aux/IAA Proteins Are Phosphorylated by Phytochrome in Vitro JO - Plant Physiol. PY - 2000 SP - 1728-1738 AU - Colón-Carmona, A. AU - Chen, D. L. AU - Yeh, K.-C. AU - Abel, S. AU - VL - 124 UR - DO - 10.1104/pp.124.4.1728 AB - Auxin/indole-3-acetic acid (Aux/IAA) genes encode short-lived transcription factors that are induced as a primary response to the plant growth hormone IAA or auxin. Gain-of-function mutations in Arabidopsis genes,SHY2/IAA3, AXR3/IAA17, andAXR2/IAA7 cause pleiotropic phenotypes consistent with enhanced auxin responses, possibly by increasing Aux/IAA protein stability. Semidominant mutations shy2-1D,shy2-2, axr3-1, and axr2-1induce ectopic light responses in dark-grown seedlings. Because genetic studies suggest that the shy2-1D andshy2-2 mutations bypass phytochrome requirement for certain aspects of photomorphogenesis, we tested whether SHY2/IAA3 and related Aux/IAA proteins interact directly with phytochrome and whether they are substrates for its protein kinase activity. Here we show that recombinant Aux/IAA proteins from Arabidopsis and pea (Pisum sativum) interact in vitro with recombinant phytochrome A from oat (Avena sativa). We further show that recombinant SHY2/IAA3, AXR3/IAA17, IAA1, IAA9, and Ps-IAA4 are phosphorylated by recombinant oat phytochrome A in vitro. Deletion analysis of Ps-IAA4 indicates that phytochrome A phosphorylation occurs on the N-terminal half of the protein. Metabolic labeling and immunoprecipitation studies with affinity-purified antibodies to IAA3 demonstrate increased in vivo steady-state levels of mutant IAA3 in shy2-2 plants and phosphorylation of the SHY2-2 protein in vivo. Phytochrome-dependent phosphorylation of Aux/IAA proteins is proposed to provide one molecular mechanism for integrating auxin and light signaling in plant development. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2253 TI - Conditional identification of phosphate-starvation-response mutants in Arabidopsis thaliana JO - Planta PY - 2000 SP - 13-22 AU - Chen, D. L. AU - Delatorre, C. A. AU - Bakker, A. AU - Abel, S. AU - VL - 211 UR - DO - 10.1007/s004250000271 AB - Plants have evolved elaborate metabolic and developmental adaptations to low phosphorus availability. Biochemical responses to phosphate limitation include increased production and secretion of phosphate-acquisition proteins such as nucleases, acid phosphatases, and high-affinity phosphate transporters. However, the signal transduction pathways that sense phosphate availability and integrate the phosphate-starvation response in plants are unknown. We have devised a screen for conditional mutants in Arabidopsis thaliana (L.) Heynh. to dissect signaling of phosphate limitation. Our genetic screen is based on the facultative ability of wild-type Arabidopsis plants to metabolize exogenous DNA when inorganic phosphate is limiting. After screening 50,000 M2 seedlings, we isolated 22 confirmed mutant lines that showed severely impaired growth on medium containing DNA as the only source of phosphorus, but which recovered on medium containing soluble inorganic phosphate. Characterization of nine such mutant lines demonstrated an inability to utilize either DNA or RNA. One mutant line, psr1 (phosphate starvation response), had significantly reduced activities of phosphate-starvation-inducible isoforms of ribonuclease and acid phosphatase under phosphate-limiting conditions. The data suggest that a subset of the selected mutations impairs the expression of more than one phosphate-starvation-inducible enzyme required for utilization of exogenous nucleic acids, and may thus affect regulatory components of a Pi starvation response pathway in higher plants. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2245 TI - Induction of an Extracellular Cyclic Nucleotide Phosphodiesterase as an Accessory Ribonucleolytic Activity during Phosphate Starvation of Cultured Tomato Cells JO - Plant Physiol. PY - 2000 SP - 543-552 AU - Abel, S. AU - Nürnberger, T. AU - Ahnert, V. AU - Krauss, G.-J. AU - Glund, K. AU - VL - 122 UR - DO - 10.1104/pp.122.2.543 AB - During growth under conditions of phosphate limitation, suspension-cultured cells of tomato (Lycopersicon esculentum Mill.) secrete phosphodiesterase activity in a similar fashion to phosphate starvation-inducible ribonuclease (RNase LE), a cyclizing endoribonuclease that generates 2′:3′-cyclic nucleoside monophosphates (NMP) as its major monomeric products (T. Nürnberger, S. Abel, W. Jost, K. Glund [1990] Plant Physiol 92: 970–976). Tomato extracellular phosphodiesterase was purified to homogeneity from the spent culture medium of phosphate-starved cells and was characterized as a cyclic nucleotide phosphodiesterase. The purified enzyme has a molecular mass of 70 kD, a pH optimum of 6.2, and an isoelectric point of 8.1. The phosphodiesterase preparation is free of any detectable deoxyribonuclease, ribonuclease, and nucleotidase activity. Tomato extracellular phosphodiesterase is insensitive to EDTA and hydrolyzes with no apparent base specificity 2′:3′-cyclic NMP to 3′-NMP and the 3′:5′-cyclic isomers to a mixture of 3′-NMP and 5′-NMP. Specific activities of the enzyme are 2-fold higher for 2′:3′-cyclic NMP than for 3′:5′-cyclic isomers. Analysis of monomeric products of sequential RNA hydrolysis with purified RNase LE, purified extracellular phosphodiesterase, and cleared −Pi culture medium as a source of 3′-nucleotidase activity indicates that cyclic nucleotide phosphodiesterase functions as an accessory ribonucleolytic activity that effectively hydrolyzes primary products of RNase LE to substrates for phosphate-starvation-inducible phosphomonoesterases. Biosynthetical labeling of cyclic nucleotide phopshodiesterase upon phosphate starvation suggests de novo synthesis and secretion of a set of nucleolytic enzymes for scavenging phosphate from extracellular RNA substrates. A2 - C1 - Molecular Signal Processing; Biochemistry of Plant Interactions ER - TY - JOUR ID - 2474 TI - Intracellular Localization of Jasmonate-Induced Proteins in Barley Leaves JO - Bot. Acta PY - 1994 SP - 333-341 AU - Hause, B. AU - zur Nieden, U. AU - Lehmann, J. AU - Wasternack, C. AU - Parthier, B. AU - VL - 107 UR - DO - 10.1111/j.1438-8677.1994.tb00804.x AB - The plant growth substance jasmonic acid and its methyl ester (JA‐Me) induce a set of proteins (jasmonate‐induced proteins, JIPs) when applied to leaf segments of barley (Hordeum vulgare L. cv. Salome). Most of these JIPs could be localized within different cell compartments by using a combination of biochemical and histochemical methods. Isolation and purification of various cell organelles of barley mesophyll cells, the separation of their proteins by one‐dimensional polyacrylamide gel electrophoresis and the identification of the major abundant JIPs by Western blot analysis, as well as the immuno‐gold labelling of JIPs in ultrathin sections were performed to localize JIPs intracellularly. JIP‐23 was found to be in vacuoles, peroxisomes, and in the granular parts of the nucleus as well as within the cytoplasm; JIP‐37 was detected in vacuoles and in the nucleoplasm; JIP‐66 is a cytosolic protein. Some less abundant JIPs were also localized within different cell compartments: JIP‐100 was found within the stromal fraction of chloroplasts; JIP‐70 is present in the peroxisome and the nucleus; JIP‐50 and JIP‐6 accumulate in vacuoles. The location of JIP‐66 and JIP‐6 confirms their possible physiological role deduced from molecular analysis of their cDNA. A2 - C1 - Molecular Signal Processing; Cell and Metabolic Biology ER - TY - JOUR ID - 2470 TI - Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression JO - Plant J. PY - 1994 SP - 421-427 AU - Abel, S. AU - Theologis, A. AU - VL - 5 UR - DO - 10.1111/j.1365-313X.1994.00421.x AB - An improved protocol is reported to isolate and transiently transform mesophyll protoplasts of Arabidopsis thaliana. Transfected leaf protoplasts support high levels of expression of the bacterial reporter gene coding for β‐glucuronidase (GUS), under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Transient expression of GUS activity was monitored spectrophotometrically and reached a maximum between 18 and 48 h after polyethylene glycol (PEG)‐mediated DNA uptake. Histochemical staining for GUS activity revealed reproducible transformation frequencies between 40 and 60%, based on the number of protoplasts survived. To demonstrate the applicability of the transient expression system, the subcellular localization of GUS proteins tagged with different nuclear polypeptides was studied in transfected mesophyll protoplasts, revealing nuclear compartmentalization of the chimeric GUS enzymes. Furthermore, Arabidopsis mesophyll protoplasts support auxin‐mediated induction of chloramphenicol acetyl‐transferase (CAT) activity when transfected with a transcriptional fusion between the CAT reporter gene and the early auxin‐inducible PS‐IAA4/5 promoter. Hence, the method allows in vivo analysis of promoter activity and subcellular localization of fusion proteins in a homologous transformation system. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2469 TI - Early auxin-induced genes encode short-lived nuclear proteins. JO - Proc. Natl. Acad. Sci. U.S.A. PY - 1994 SP - 326-330 AU - Abel, S. AU - Oeller, P. W. AU - Theologis, A. AU - VL - 91 UR - DO - 10.1073/pnas.91.1.326 AB - The plant growth hormone indoleacetic acid (IAA) transcriptionally activates gene expression in plants. Some of the genes whose expression is induced by IAA encode a family of proteins in pea (PS-IAA4 and PS-IAA6) and Arabidopsis (IAA1 and IAA2) that contain putative nuclear localization signals that direct a beta-glucuronidase reporter protein into the nucleus. Pulse-chase and immunoprecipitation experiments have defined the t1/2 of the PS-IAA4 and PS-IAA6 proteins to be 8 and 6 min, respectively. Their most prominent feature is the presence of a beta alpha alpha motif similar to the beta-sheet DNA-binding domain found in prokaryotic repressors of the Arc family. Based on these data, we suggest that plant tissues express short-lived nuclear proteins as a primary response to IAA. We propose that these proteins act as activators or repressors of genes responsible for mediating the various auxin responses. A2 - C1 - Molecular Signal Processing ER -