TY - JOUR ID - 203 TI - The activation of iron deficiency responses of grapevine rootstocks is dependent to the availability of the nitrogen forms JO - BMC Plant Biol. PY - 2024 SP - 218 AU - Khalil, S. AU - Strah, R. AU - Lodovici, A. AU - Vojta, P. AU - Berardinis, F. D. AU - Ziegler, J. AU - Novak, M. P. AU - Zanin, L. AU - Tomasi, N. AU - Forneck, A. AU - Griesser, M. AU - VL - 24 UR - https://doi.org/10.1186/s12870-024-04906-y DO - 10.1186/s12870-024-04906-y AB - Background  In viticulture, iron (Fe) chlorosis is a common abiotic stress that impairs plant development and leads to yield and quality losses. Under low availability of the metal, the applied N form (nitrate and ammonium) can play a role in promoting or mitigating Fe deficiency stresses. However, the processes involved are not clear in grapevine. Therefore, the aim of this study was to investigate the response of two grapevine rootstocks to the interaction between N forms and Fe uptake. This process was evaluated in a hydroponic experiment using two ungrafted grapevine rootstocks Fercal (Vitis berlandieri x V. vinifera) tolerant to deficiency induced Fe chlorosis and Couderc 3309 (V. riparia x V. rupestris) susceptible to deficiency induced Fe chlorosis. Results  The results could differentiate Fe deficiency effects, N-forms effects, and rootstock effects. Interveinal chlorosis of young leaves appeared earlier on 3309 C from the second week of treatment with NO3−/NH4+ (1:0)/-Fe, while Fercal leaves showed less severe symptoms after four weeks of treatment, corresponding to decreased chlorophyll concentrations lowered by 75% in 3309 C and 57% in Fercal. Ferric chelate reductase (FCR) activity was by trend enhanced under Fe deficiency in Fercal with both N combinations, whereas 3309 C showed an increase in FCR activity under Fe deficiency only with NO3−/NH4+ (1:1) treatment. With the transcriptome analysis, Gene Ontology (GO) revealed multiple biological processes and molecular functions that were significantly regulated in grapevine rootstocks under Fe-deficient conditions, with more genes regulated in Fercal responses, especially when both forms of N were supplied. Furthermore, the expression of genes involved in the auxin and abscisic acid metabolic pathways was markedly increased by the equal supply of both forms of N under Fe deficiency conditions. In addition, changes in the expression of genes related to Fe uptake, regulation, and transport reflected the different responses of the two grapevine rootstocks to different N forms. Conclusions  Results show a clear contribution of N forms to the response of the two grapevine rootstocks under Fe deficiency, highlighting the importance of providing both N forms (nitrate and ammonium) in an appropriate ratio in order to ease the rootstock responses to Fe deficiency. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 195 TI - A CYBDOM protein impacts iron homeostasis and primary root growth under phosphate deficiency in Arabidopsis JO - Nat. Commun. PY - 2024 SP - 423 AU - Clúa, J. AU - Montpetit, J. AU - Jimenez-Sandoval, P. AU - Naumann, C. AU - Santiago, J. AU - Poirier, Y. AU - VL - 15 UR - https://doi.org/10.1038/s41467-023-43911-x DO - 10.1038/s41467-023-43911-x AB - Arabidopsis primary root growth response to phosphate (Pi) deficiency is mainly controlled by changes in apoplastic iron (Fe). Upon Pi deficiency, apoplastic Fe deposition in the root apical meristem activates pathways leading to the arrest of meristem maintenance and inhibition of cell elongation. Here, we report that a member of the uncharacterized cytochrome b561 and DOMON domain (CYBDOM) protein family, named CRR, promotes iron reduction in an ascorbate-dependent manner and controls apoplastic iron deposition. Under low Pi, the crr mutant shows an enhanced reduction of primary root growth associated with increased apoplastic Fe in the root meristem and a reduction in meristematic cell division. Conversely, CRR overexpression abolishes apoplastic Fe deposition rendering primary root growth insensitive to low Pi. The crr single mutant and crr hyp1 double mutant, harboring a null allele in another member of the CYDOM family, shows increased tolerance to high-Fe stress upon germination and seedling growth. Conversely, CRR overexpression is associated with increased uptake and translocation of Fe to the shoot and results in plants highly sensitive to Fe excess. Our results identify a ferric reductase implicated in Fe homeostasis and developmental responses to abiotic stress, and reveal a biological role for CYBDOM proteins in plants. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 265 TI - The secreted PAMP-induced peptide StPIP1_1 activates immune responses in potato JO - Sci. Rep. PY - 2023 SP - 20534 AU - Nietzschmann, L. AU - Smolka, U. AU - Perino, E. H. B. AU - Gorzolka, K. AU - Stamm, G. AU - Marillonnet, S. AU - Bürstenbinder, K. AU - Rosahl, S. AU - VL - 13 UR - https://doi.org/10.1038/s41598-023-47648-x DO - 10.1038/s41598-023-47648-x AB - Treatment of potato plants with the pathogen-associated molecular pattern Pep-13 leads to the activation of more than 1200 genes. One of these, StPIP1_1, encodes a protein of 76 amino acids with sequence homology to PAMP-induced secreted peptides (PIPs) from Arabidopsis thaliana. Expression of StPIP1_1 is also induced in response to infection with Phytophthora infestans, the causal agent of late blight disease. Apoplastic localization of StPIP1_1-mCherry fusion proteins is dependent on the presence of the predicted signal peptide. A synthetic peptide corresponding to the last 13 amino acids of StPIP1_1 elicits the expression of the StPIP1_1 gene itself, as well as that of pathogenesis related genes. The oxidative burst induced by exogenously applied StPIP1_1 peptide in potato leaf disks is dependent on functional StSERK3A/B, suggesting that StPIP1_1 perception occurs via a receptor complex involving the co-receptor StSERK3A/B. Moreover, StPIP1_1 induces expression of FRK1 in Arabidopsis in an RLK7-dependent manner. Expression of an RLK from potato with high sequence homology to AtRLK7 is induced by StPIP1_1, by Pep-13 and in response to infection with P. infestans. These observations are consistent with the hypothesis that, upon secretion, StPIP1_1 acts as an endogenous peptide required for amplification of the defense response. A2 - C1 - Molecular Signal Processing; Biochemistry of Plant Interactions ER - TY - JOUR ID - 264 TI - Endoplasmic reticulum calnexins participate in the primary root growth response to phosphate deficiency JO - Plant Physiol. PY - 2023 SP - 1719-1733 AU - Montpetit, J. AU - Clúa, J. AU - Hsieh, Y.-F. AU - Vogiatzaki, E. AU - Müller, J. AU - Abel, S. AU - Strasser, R. AU - Poirier, Y. AU - VL - 191 UR - https://doi.org/10.1093/plphys/kiac595 DO - 10.1093/plphys/kiac595 AB - Accumulation of incompletely folded proteins in the endoplasmic reticulum (ER) leads to ER stress, activates ER protein degradation pathways, and upregulates genes involved in protein folding. This process is known as the unfolded protein response (UPR). The role of ER protein folding in plant responses to nutrient deficiencies is unclear. We analyzed Arabidopsis (Arabidopsis thaliana) mutants affected in ER protein quality control and established that both CALNEXIN (CNX) genes function in the primary root’s response to phosphate (Pi) deficiency. CNX1 and CNX2 are homologous ER lectins promoting protein folding of N-glycosylated proteins via the recognition of the GlcMan9GlcNAc2 glycan. Growth of cnx1-1 and cnx2-2 single mutants was similar to that of the wild type under high and low Pi conditions, but the cnx1-1 cnx2-2 double mutant showed decreased primary root growth under low Pi conditions due to reduced meristematic cell division. This phenotype was specific to Pi deficiency; the double mutant responded normally to osmotic and salt stress. Expression of CNX2 mutated in amino acids involved in binding the GlcMan9GlcNAc2 glycan failed to complement the cnx1-1 cnx2-2 mutant. The root growth phenotype was Fe dependent and was associated with root apoplastic Fe accumulation. Two genes involved in Fe-dependent inhibition of primary root growth under Pi deficiency, the ferroxidase LOW PHOSPHATE 1 (LPR1) and P5-type ATPase PLEIOTROPIC DRUG RESISTANCE 2 (PDR2) were epistatic to CNX1/CNX2. Overexpressing PDR2 failed to complement the cnx1-1 cnx2-2 root phenotype. The cnx1-1 cnx2-2 mutant showed no evidence of UPR activation, indicating a limited effect on ER protein folding. CNX might process a set of N-glycosylated proteins specifically involved in the response to Pi deficiency. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 241 TI - Molecular understanding of ACE-2 and HLA-conferred differential susceptibility to COVID-19: Host-directed insights opening new windows in COVID-19 therapeutics JO - Journal of Clinical Medicine PY - 2023 SP - 2645 AU - Haq, I. U. AU - Krukiewicz, K. AU - Tayyab, H. AU - Khan, I. AU - Khan, M. AU - Yahya, G. AU - Cavalu, S. AU - VL - 12 UR - https://doi.org/10.3390/jcm12072645 DO - 10.3390/jcm12072645 AB - The genetic variants of HLAs (human leukocyte antigens) play a crucial role in the virus–host interaction and pathology of COVID-19. The genetic variants of HLAs not only influence T cell immune responses but also B cell immune responses by presenting a variety of peptide fragments of invading pathogens. Peptide cocktail vaccines produced by using various conserved HLA-A2 epitopes provoke substantial specific CD8+ T cell responses in experimental animals. The HLA profiles vary among individuals and trigger different T cell-mediated immune responses in COVID-19 infections. Those with HLA-C*01 and HLA-B*44 are highly susceptible to the disease. However, HLA-A*02:01, HLA-DR*03:01, and HLA-Cw*15:02 alleles show resistance to SARS infection. Understanding the genetic association of HLA with COVID-19 susceptibility and severity is important because it can help in studying the transmission of COVID-19 and its physiopathogenesis. The HLA-C*01 and B*44 allele pathways can be studied to gain insight into disease transmission and physiopathogenesis. Therefore, integrating HLA testing is suggested in the ongoing pandemic, which will help in the rapid identification of highly susceptible populations worldwide and possibly acclimate vaccine development. Therefore, understanding the correlation between HLA and SARS-CoV-2 is critical in opening new insights into COVID-19 therapeutics, based on previous studies conducted. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 290 TI - Improved 13C metabolic flux analysis in Escherichia coli metabolism: application of a high-resolution MS (GC–EI–QTOF) for comprehensive assessment of MS/MS fragments JO - Journal of Industrial Microbiology and Biotechnology PY - 2023 SP - kuad039 AU - Richter, C. AU - Grafahrend-Belau, E. AU - Ziegler, J. AU - Raorane, M. L. AU - Junker, B. H. AU - VL - 50 UR - https://doi.org/10.1093/jimb/kuad039 DO - 10.1093/jimb/kuad039 AB - Gas chromatography–tandem mass spectrometry with electron ionization (GC–EI–MS/MS) provides rich information on stable-isotope labeling for 13C-metabolic flux analysis (13C-MFA). To pave the way for the routine application of tandem MS data for metabolic flux quantification, we aimed to compile a comprehensive library of GC–EI–MS/MS fragments of tert-butyldimethylsilyl (TBDMS) derivatized proteinogenic amino acids. First, we established an analytical workflow that combines high-resolution gas chromatography-quadrupole time-of-flight mass spectrometry and fully 13C-labeled biomass to identify and structurally elucidate tandem MS amino acid fragments. Application of the high-mass accuracy MS procedure resulted into the identification of 129 validated precursor–product ion pairs of 13 amino acids with 30 fragments being accepted for 13C-MFA. The practical benefit of the novel tandem MS data was demonstrated by a proof–of–concept study, which confirmed the importance of the compiled library for high-resolution 13C-MFA. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 278 TI - Shuffled ATG8 interacting motifs form an ancestral bridge between UFMylation and autophagy JO - EMBO J. PY - 2023 SP - e112053 AU - Picchianti, L. AU - Sanchez de Medina Hernandez, V. AU - Zhan, N. AU - Irwin, N. A. AU - Groh, R. AU - Stephani, M. AU - Hornegger, H. AU - Beveridge, R. AU - Sawa‐Makarska, J. AU - Lendl, T. AU - Grujic, N. AU - Naumann, C. AU - Martens, S. AU - Richards, T. A. AU - Clausen, T. AU - Ramundo, S. AU - Karagöz, G. E. AU - Dagdas, Y. AU - VL - 42 UR - https://doi.org/10.15252/embj.2022112053 DO - 10.15252/embj.2022112053 AB - UFMylation involves the covalent modification of substrate proteins with UFM1 (Ubiquitin-fold modifier 1) and is important for maintaining ER homeostasis. Stalled translation triggers the UFMylation of ER-bound ribosomes and activates C53-mediated autophagy to clear toxic polypeptides. C53 contains noncanonical shuffled ATG8-interacting motifs (sAIMs) that are essential for ATG8 interaction and autophagy initiation. However, the mechanistic basis of sAIM-mediated ATG8 interaction remains unknown. Here, we show that C53 and sAIMs are conserved across eukaryotes but secondarily lost in fungi and various algal lineages. Biochemical assays showed that the unicellular alga Chlamydomonas reinhardtii has a functional UFMylation pathway, refuting the assumption that UFMylation is linked to multicellularity. Comparative structural analyses revealed that both UFM1 and ATG8 bind sAIMs in C53, but in a distinct way. Conversion of sAIMs into canonical AIMs impaired binding of C53 to UFM1, while strengthening ATG8 binding. Increased ATG8 binding led to the autoactivation of the C53 pathway and sensitization of Arabidopsis thaliana to ER stress. Altogether, our findings reveal an ancestral role of sAIMs in UFMylation-dependent fine-tuning of C53-mediated autophagy activation. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 274 TI - Light exposure of roots in aeroponics enhances the accumulation of phytochemicals in aboveground parts of the medicinal plants Artemisia annua and Hypericum perforatum JO - Front. Plant Sci. PY - 2023 SP - 1079656 AU - Paponov, M. AU - Ziegler, J. AU - Paponov, I. A. AU - VL - 14 UR - https://doi.org/10.3389/fpls.2023.1079656 DO - 10.3389/fpls.2023.1079656 AB - Light acts as a trigger to enhance the accumulation of secondary compounds in the aboveground part of plants; however, whether a similar triggering effect occurs in roots is unclear. Using an aeroponic setup, we investigated the effect of long-term exposure of roots to LED lighting of different wavelengths on the growth and phytochemical composition of two high-value medicinal plants, Artemisia annua and Hypericum perforatum. In A. annua, root exposure to white, blue, and red light enhanced the accumulation of artemisinin in the shoots by 2.3-, 2.5-, and 1.9-fold, respectively. In H. perforatum, root exposure to white, blue, red, and green light enhanced the accumulation of coumaroylquinic acid in leaves by 89, 65, 84, and 74%, respectively. Root lighting also increased flavonol concentrations. In contrast to its effects in the shoots, root illumination did not change phytochemical composition in the roots or root exudates. Thus, root illumination induces a systemic response, resulting in modulation of the phytochemical composition in distal tissues remote from the light exposure site. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 273 TI - Heterogeneous nutrient supply modulates root exudation and accumulation of medicinally valuable compounds in Artemisia annua and Hypericum perforatum JO - Front. Plant Sci. PY - 2023 SP - 1174151 AU - Paponov, M. AU - Flate, J. AU - Ziegler, J. AU - Lillo, C. AU - Paponov, I. A. AU - VL - 14 UR - https://doi.org/10.3389/fpls.2023.1174151 DO - 10.3389/fpls.2023.1174151 AB - Plants have evolved complex mechanisms to adapt to nutrient-deficient environments, including stimulating lateral root proliferation into local soil patches with high nutrient content in response to heterogeneous nutrient distribution. Despite the widespread occurrence of this phenomenon in soil, the effect of heterogeneous nutrient distribution on the accumulation of secondary compounds in plant biomass and their exudation by roots remains largely unknown. This study aims to fill this critical knowledge gap by investigating how deficiency and unequal distributions of nitrogen (N), phosphorus (P), and iron (Fe) affect plant growth and accumulation of the antimalarial drug artemisinin (AN) in leaves and roots of Artemisia annua, as well as AN exudation by roots. Heterogeneous N and P supplies strongly increased root exudation of AN in half of a split-root system exposed to nutrient deficiency. By contrast, exposure to a homogeneous nitrate and phosphate deficiency did not modulate root exudation of AN. This indicates that a combination of local and systemic signals, reflecting low and high nutritional statuses, respectively, were required to enhance AN exudation. This exudation response was independent of the regulation of root hair formation, which was predominantly modulated by the local signal. In contrast to the heterogeneous supply of N and P, heterogeneous Fe supply did not modulate AN root exudation but increased AN accumulation in locally Fe-deficient roots. No modulation of nutrient supply significantly changed the accumulation of AN in A. annua leaves. The impact of a heterogeneous nitrate supply on growth and phytochemical composition was also investigated in Hypericum perforatum plants. Unlike in A. annue, the uneven N supply did not significantly influence the exudation of secondary compounds in the roots of H. perforatum. However, it did enhance the accumulation of several biologically active compounds, such as hypericin, catechin, and rutin isomers, in the leaves of H. perforatum. We propose that the capacity of plants to induce the accumulation and/or differential exudation of secondary compounds under heterogeneous nutrient supply is both species- and compound-specific. The ability to differentially exude AN may contribute to A. annua’s adaptation to nutrient disturbances and modulate allelopathic and symbiotic interactions in the rhizosphere. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 225 TI - Impact of in vitro phytohormone treatments on the metabolome of the leafy liverwort Radula complanata (L.) Dumort JO - Metabolomics PY - 2023 SP - 17 AU - Blatt-Janmaat, K. AU - Neumann, S. AU - Schmidt, F. AU - Ziegler, J. AU - Qu, Y. AU - Peters, K. AU - VL - 19 UR - https://doi.org/10.1007/s11306-023-01979-y DO - 10.1007/s11306-023-01979-y AB - Introduction Liverworts are a group of non-vascular plants that possess unique metabolism not found in other plants. Many liverwort metabolites have interesting structural and biochemical characteristics, however the fluctuations of these metabolites in response to stressors is largely unknown. Objectives To investigate the metabolic stress-response of the leafy liverwort Radula complanata. Methods Five phytohormones were applied exogenously to in vitro cultured R. complanata and an untargeted metabolomic analysis was conducted. Compound classification and identification was performed with CANOPUS and SIRIUS while statistical analyses including PCA, ANOVA, and variable selection using BORUTA were conducted to identify metabolic shifts.Results It was found that R. complanata was predominantly composed of carboxylic acids and derivatives, followed by benzene and substituted derivatives, fatty acyls, organooxygen compounds, prenol lipids, and flavonoids. The PCA revealed that samples grouped based on the type of hormone applied, and the variable selection using BORUTA (Random Forest) revealed 71 identified and/or classified features that fluctuated with phytohormone application. The stress-response treatments largely reduced the production of the selected primary metabolites while the growth treatments resulted in increased production of these compounds. 4-(3-Methyl-2-butenyl)-5-phenethylbenzene-1,3-diol was identified as a biomarker for the growth treatments while GDP-hexose was identified as a biomarker for the stress-response treatments. Conclusion Exogenous phytohormone application caused clear metabolic shifts in Radula complanata that deviate from the responses of vascular plants. Further identification of the selected metabolite features can reveal metabolic biomarkers unique to liverworts and provide more insight into liverwort stress responses. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 224 TI - Host tree and geography induce metabolic shifts in the epiphytic liverwort Radula complanata JO - Plants PY - 2023 SP - 571 AU - Blatt-Janmaat, K. L. AU - Neumann, S. AU - Ziegler, J. AU - Peters, K. AU - VL - 12 UR - https://doi.org/10.3390/plants12030571 DO - 10.3390/plants12030571 AB - Bryophytes are prolific producers of unique, specialized metabolites that are not found in other plants. As many of these unique natural products are potentially interesting, for example, pharmacological use, variations in the production regarding ecological or environmental conditions have not often been investigated. Here, we investigate metabolic shifts in the epiphytic Radula complanata L. (Dumort) with regard to different environmental conditions and the type of phorophyte (host tree). Plant material was harvested from three different locations in Sweden, Germany, and Canada and subjected to untargeted liquid chromatography high-resolution mass-spectrometry (UPLC/ESI-QTOF-MS) and data-dependent acquisition (DDA-MS). Using multivariate statistics, variable selection methods, in silico compound identification, and compound classification, a large amount of variation (39%) in the metabolite profiles was attributed to the type of host tree and 25% to differences in environmental conditions. We identified 55 compounds to vary significantly depending on the host tree (36 on the family level) and 23 compounds to characterize R. complanata in different environments. Taken together, we found metabolic shifts mainly in primary metabolites that were associated with the drought response to different humidity levels. The metabolic shifts were highly specific to the host tree, including mostly specialized metabolites suggesting high levels of ecological interaction. As R. complanata is a widely distributed generalist species, we found it to flexibly adapt its metabolome according to different conditions. We found metabolic composition to also mirror the constitution of the habitat, which makes it interesting for conservation measures. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 220 TI - An LRR receptor kinase controls ABC transporter substrate preferences during plant growth-defense decisions JO - Curr. Biol. PY - 2023 SP - 2008-2023 AU - Aryal, B. AU - Xia, J. AU - Hu, Z. AU - Stumpe, M. AU - Tsering, T. AU - Liu, J. AU - Huynh, J. AU - Fukao, Y. AU - Glöckner, N. AU - Huang, H.-Y. AU - Sancho-Andrés, G. AU - Pakula, K. AU - Ziegler, J. AU - Gorzolka, K. AU - Zwiewka, M. AU - Nodzynski, T. AU - Harter, K. AU - Sánchez-Rodríguez, C. AU - Jasiński, M. AU - Rosahl, S. AU - Geisler, M. M. AU - VL - 33 UR - https://doi.org/10.1016/j.cub.2023.04.029 DO - 10.1016/j.cub.2023.04.029 AB - The exporter of the auxin precursor indole-3-butyric acid (IBA), ABCG36/PDR8/PEN3, from the model plant Arabidopsis has recently been proposed to also function in the transport of the phytoalexin camalexin. Based on these bonafide substrates, it has been suggested that ABCG36 functions at the interface between growth and defense. Here, we provide evidence that ABCG36 catalyzes the direct, ATP-dependent export of camalexin across the plasma membrane. We identify the leucine-rich repeat receptor kinase, QIAN SHOU KINASE1 (QSK1), as a functional kinase that physically interacts with and phosphorylates ABCG36. Phosphorylation of ABCG36 by QSK1 unilaterally represses IBA export, allowing camalexin export by ABCG36 conferring pathogen resistance. As a consequence, phospho-dead mutants of ABCG36, as well as qsk1 and abcg36 alleles, are hypersensitive to infection with the root pathogen Fusarium oxysporum, caused by elevated fungal progression. Our findings indicate a direct regulatory circuit between a receptor kinase and an ABC transporter that functions to control transporter substrate preference during plant growth and defense balance decisions. A2 - C1 - Biochemistry of Plant Interactions; Molecular Signal Processing ER - TY - JOUR ID - 217 TI - Changing turn-over rates regulate abundance of tryptophan, GS biosynthesis, IAA transport and photosynthesis proteins in Arabidopsis growth defense transitions JO - BMC Biol. PY - 2023 SP - 249 AU - Abukhalaf, M. AU - Proksch, C. AU - Thieme, D. AU - Ziegler, J. AU - Hoehenwarter, W. AU - VL - 21 UR - https://doi.org/10.1186/s12915-023-01739-3 DO - 10.1186/s12915-023-01739-3 AB - Background Shifts in dynamic equilibria of the abundance of cellular molecules in plant-pathogen interactions need further exploration. We induced PTI in optimally growing Arabidopsis thaliana seedlings for 16 h, returning them to growth conditions for another 16 h. Methods Turn-over and abundance of 99 flg22 responding proteins were measured chronologically using a stable heavy nitrogen isotope partial labeling strategy and targeted liquid chromatography coupled to mass spectrometry (PRM LC–MS). These experiments were complemented by measurements of mRNA and phytohormone levels. Results Changes in synthesis and degradation rate constants (Ks and Kd) regulated tryptophane and glucosinolate, IAA transport, and photosynthesis-associated protein (PAP) homeostasis in growth/PTI transitions independently of mRNA levels. Ks values increased after elicitation while protein and mRNA levels became uncorrelated. mRNA returned to pre-elicitation levels, yet protein abundance remained at PTI levels even 16 h after media exchange, indicating protein levels were robust and unresponsive to transition back to growth. The abundance of 23 PAPs including FERREDOXIN-NADP( +)-OXIDOREDUCTASE (FNR1) decreased 16 h after PAMP exposure, their depletion was nearly abolished in the myc234 mutant. FNR1 Kd increased as mRNA levels decreased early in PTI, its Ks decreased in prolonged PTI. FNR1 Kd was lower in myc234, mRNA levels decreased as in wild type. Conclusions Protein Kd and Ks values change in response to flg22 exposure and constitute an additional layer of protein abundance regulation in growth defense transitions next to changes in mRNA levels. Our results suggest photosystem remodeling in PTI to direct electron flow away from the photosynthetic carbon reaction towards ROS production as an active defense mechanism controlled post-transcriptionally and by MYC2 and homologs. Target proteins accumulated later and PAP and auxin/IAA depletion was repressed in myc234 indicating a positive effect of the transcription factors in the establishment of PTI. A2 - C1 - Biochemistry of Plant Interactions; Molecular Signal Processing ER - TY - JOUR ID - 396 TI - Native protein purification of ferroxidase LPR1 from leaf extracts of a transgenic Arabidopsis thaliana line JO - STAR Protocols PY - 2022 SP - 101733 AU - Tang, N. AU - Naumann, C. AU - VL - 3 UR - https://doi.org/10.1016/j.xpro.2022.101733 DO - 10.1016/j.xpro.2022.101733 AB - (LOW PHOSPHATE ROOT 1), a bacterial-type plant ferroxidase, is crucial for local root phosphate (Pi) sensing. Here, we present a detailed protocol for native (tag-free) protein purification of LPR1 from leaf extracts by differential ammonium sulfate precipitation, size exclusion, and cation exchange chromatography of a transgenic Arabidopsis thaliana line overexpressing LPR1. We outline steps for LPR1 purification tracking via immune blot analysis and ferroxidase activity assay. The protocol yields highly pure and active LPR1 protein for biochemical analysis. For complete details on the use and execution of this protocol, please refer to Naumann et al. (2022) A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 382 TI - Mechanism of chorismate dehydratase MqnA, the first enzyme of the futalosine pathway, proceeds via substrate-assisted catalysis JO - J. Biol. Chem. PY - 2022 SP - 102601 AU - Prasad, A. AU - Breithaupt, C. AU - Nguyen, D.-A. AU - Lilie, H. AU - Ziegler, J. AU - Stubbs, M. T. AU - VL - 298 UR - https://doi.org/10.1016/j.jbc.2022.102601 DO - 10.1016/j.jbc.2022.102601 AB - MqnA, the only chorismate dehydratase known so far, catalyzes the initial step in the biosynthesis of menaquinone via the futalosine pathway. Details of the MqnA reaction mechanism remain unclear. Here, we present crystal structures of Streptomyces coelicolor MqnA and its active site mutants in complex with chorismate and the product 3-enolpyruvyl-benzoate, produced during heterologous expression in Escherichia coli. Together with activity studies, our data are in line with dehydration proceeding via substrate assisted catalysis, with the enol pyruvyl group of chorismate acting as catalytic base. Surprisingly, structures of the mutant Asn17Asp with copurified ligand suggest that the enzyme converts to a hydrolase by serendipitous positioning of the carboxyl group. All complex structures presented here exhibit a closed Venus flytrap fold, with the enzyme exploiting the characteristic ligand binding properties of the fold for specific substrate binding and catalysis. The conformational rearrangements that facilitate complete burial of substrate/product, with accompanying topological changes to the enzyme surface, could foster substrate channeling within the biosynthetic pathway. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 376 TI - Bacterial-type ferroxidase tunes iron-dependent phosphate sensing during Arabidopsis root development JO - Curr. Biol. PY - 2022 SP - 2189-2205 AU - Naumann, C. AU - Heisters, M. AU - Brandt, W. AU - Janitza, P. AU - Alfs, C. AU - Tang, N. AU - Toto Nienguesso, A. AU - Ziegler, J. AU - Imre, R. AU - Mechtler, K. AU - Dagdas, Y. AU - Hoehenwarter, W. AU - Sawers, G. AU - Quint, M. AU - Abel, S. AU - VL - 32 UR - https://doi.org/10.1016/j.cub.2022.04.005 DO - 10.1016/j.cub.2022.04.005 AB - Access to inorganic phosphate (Pi), a principal intermediate of energy and nucleotide metabolism, profoundly affects cellular activities and plant performance. In most soils, antagonistic Pi-metal interactions restrict Pi bioavailability, which guides local root development to maximize Pi interception. Growing root tips scout the essential but immobile mineral nutrient; however, the mechanisms monitoring external Pi sta-tus are unknown. Here, we show that Arabidopsis LOW PHOSPHATE ROOT 1 (LPR1), one key determinant of Fe-dependent Pi sensing in root meristems, encodes a novel ferroxidase of high substrate specificity and affinity (apparent KM ∼2 μmM Fe2+). LPR1 typifies an ancient, Fe-oxidizing multicopper protein family that evolved early upon bacterial land colonization. The ancestor of streptophyte algae and embryophytes (land plants) acquired LPR1-type ferroxidase from soil bacteria via horizontal gene transfer, a hypothesis supported by phylogenomics, homology modeling, and biochemistry. Our molecular and kinetic data on LPR1 regulation indicate that Pi-dependent Fe substrate availability determines LPR1 activity and function. Guided by the metabolic lifestyle of extant sister bacterial genera, we propose that Arabidopsis LPR1 monitors subtle concentration differentials of external Fe availability as a Pi-dependent cue to adjust root meristem maintenance via Fe redox signaling and cell wall modification. We further hypothesize that the acquisition of bacterial LPR1-type ferroxidase by embryophyte progenitors facilitated the evolution of local Pi sensing and acquisition during plant terrestrialization. A2 - C1 - Bioorganic Chemistry; Molecular Signal Processing ER - TY - JOUR ID - 531 TI - Aminosäurennachweis in geringsten ProbenmengenBestimmung von Hypoglycin A JO - Wiley Analytical Science PY - 2021 SP - AU - Ziegler, J. AU - Bochnia, M. AU - Zeyner, A. AU - VL - UR - https://analyticalscience.wiley.com/do/10.1002/was.000600226/full/ AB - A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 431 TI - Hypoglycin A in cow’s milk—A pilot study JO - Toxins PY - 2021 SP - 381 AU - Bochnia, M. AU - Ziegler, J. AU - Glatter, M. AU - Zeyner, A. AU - VL - 13 UR - https://www.mdpi.com/2072-6651/13/6/381 DO - 10.3390/toxins13060381 AB - Hypoglycin A (HGA) originating from soapberry fruits (litchi, and ackee) seeds or seedlings from the sycamore maple (SM) tree (related to Sapindaceae) may cause Jamaican vomiting sickness in humans and atypical myopathy in horses and ruminants. A possible transfer into dairy cow’s milk cannot be ruled out since the literature has revealed HGA in the milk of mares and in the offal of captured deer following HGA intoxication. From a study, carried out for another purpose, bulk raw milk samples from four randomly selected dairy farms were available. The cows were pastured in the daytime. A sycamore maple tree was found on the pasture of farm No. 1 only. Bulk milk from the individual tank or milk filling station was sampled in parallels and analyzed for HGA by LC-ESI-MS/MS. Measurable concentrations of HGA occurred only in milk from farm No. 1 and amounted to 120 and 489 nmol/L. Despite low and very variable HGA concentrations, the results indicate that the ingested toxin, once eaten, is transferred into the milk. However, it is unknown how much HGA the individual cow ingested during grazing and what amount was transferred into the bulk milk samples. As a prerequisite for a possible future safety assessment, carry-over studies are needed. Furthermore, the toxins’ stability during milk processing should also be investigated as well. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 466 TI - Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition) JO - Autophagy PY - 2021 SP - 1-382 AU - Klionsky, D. J. AU - Abdel-Aziz, A. K. AU - Abdelfatah, S. AU - Abdellatif, M. AU - Abdoli, A. AU - Abel, S. AU - Naumann, C. AU - et al., . AU - VL - 17 UR - DO - 10.1080/15548627.2020.1797280 AB - In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis  updated  guidelines  for  monitoring  autophagy  in  different  organisms.  Despite  numerous  reviews, there  continues  to  be  confusion  regarding  acceptable  methods  to  evaluate  autophagy,  especially  in multicellular  eukaryotes.  Here,  we  present  a  set  of  guidelines  for  investigators  to  select  and  interpret methods  to  examine  autophagy  and  related  processes,  and  for  reviewers  to  provide  realistic  and reasonable  critiques  of  reports  that  are  focused  on  these  processes.  These  guidelines  are  not  meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being  asked  and  the  system  being  used.  Moreover,  no  individual  assay  is  perfect  for  every situation, calling  for  the  use  of  multiple  techniques  to  properly  monitor  autophagy  in  each  experimental  setting. Finally,  several  core  components  of  the  autophagy  machinery  have  been  implicated  in  distinct  auto-phagic  processes  (canonical  and  noncanonical  autophagy),  implying  that  genetic  approaches  to  block autophagy  should  rely  on  targeting  two  or  more  autophagy-related  genes  that  ideally  participate  in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation  in  the  field. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 464 TI - Free amino acid contents of selected Ethiopian plant and fungi species: a search for alternative natural free amino acid sources for cosmeceutical applications JO - Amino Acids PY - 2021 SP - 1105-1122 AU - Kahsay, B. N. AU - Ziegler, J. AU - Imming, P. AU - Gebre-Mariam, T. AU - Neubert, R. H. H. AU - Moeller, L. AU - VL - 53 UR - https://link.springer.com/article/10.1007/s00726-021-03008-5 DO - 10.1007/s00726-021-03008-5 AB - Free amino acids (FAAs), the major constituents of the natural moisturizing factor (NMF), are very important for maintaining the moisture balance of human skin and their deficiency results in dry skin conditions. There is a great interest in the identification and use of nature-based sources of these molecules for such cosmeceutical applications. The objective of the present study was, therefore, to investigate the FAA contents of selected Ethiopian plant and fungi species; and select the best sources so as to use them for the stated purpose. About 59 different plant species and oyster mushroom were included in the study and the concentrations of 27 FAAs were analyzed. Each sample was collected, lyophilized, extracted using aqueous solvent, derivatized with Fluorenylmethoxycarbonyl chloride (Fmoc-Cl) prior to solid-phase extraction and quantified using Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometric (LC-ESI–MS/MS) system. All the 27 FAAs were detected in most of the samples. The dominant FAAs that are part of the NMF were found at sufficiently high concentration in the mushroom and some of the plants. This indicates that FAAs that could be included in the preparations for the management of dry skin condition can be obtained from a single natural resource and the use of these resources for the specified purpose have both economic and therapeutic advantage in addition to fulfilling customer needs. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 455 TI - A microbiota–root–shoot circuit favours Arabidopsis growth over defence under suboptimal light JO - Nat. Plants PY - 2021 SP - 1078–1092 AU - Hou, S. AU - Thiergart, T. AU - Vannier, N. AU - Mesny, F. AU - Ziegler, J. AU - Pickel, B. AU - Hacquard, S. AU - VL - 7 UR - https://www.nature.com/articles/s41477-021-00956-4 DO - 10.1038/s41477-021-00956-4 AB - AbstractBidirectional root–shoot signalling is probably key in orchestrating stress responses and ensuring plant survival. Here, we show that Arabidopsis thaliana responses to microbial root commensals and light are interconnected along a microbiota–root–shoot axis. Microbiota and light manipulation experiments in a gnotobiotic plant system reveal that low photosynthetically active radiation perceived by leaves induces long-distance modulation of root bacterial communities but not fungal or oomycete communities. Reciprocally, microbial commensals alleviate plant growth deficiency under low photosynthetically active radiation. This growth rescue was associated with reduced microbiota-induced aboveground defence responses and altered resistance to foliar pathogens compared with the control light condition. Inspection of a set of A. thaliana mutants reveals that this microbiota- and light-dependent growth–defence trade-off is directly explained by belowground bacterial community composition and requires the host transcriptional regulator MYC2. Our work indicates that aboveground stress responses in plants can be modulated by signals from microbial root commensals. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 435 TI - Modulation of phosphate deficiency-induced metabolic changes by iron availability in Arabidopsis thaliana JO - Int. J. Mol. Sci. PY - 2021 SP - 7609 AU - Chutia, R. AU - Scharfenberg, S. AU - Neumann, S. AU - Abel, S. AU - Ziegler, J. AU - VL - 22 UR - https://doi.org/10.3390/ijms22147609 DO - 10.3390/ijms22147609 AB - Concurrent suboptimal supply of several nutrients requires the coordination of nutrient-specific transcriptional, phenotypic, and metabolic changes in plants in order to optimize growth and development in most agricultural and natural ecosystems. Phosphate (Pi) and iron (Fe) deficiency induce overlapping but mostly opposing transcriptional and root growth responses in Arabidopsis thaliana. On the metabolite level, Pi deficiency negatively modulates Fe deficiency-induced coumarin accumulation, which is controlled by Fe as well as Pi deficiency response regulators. Here, we report the impact of Fe availability on seedling growth under Pi limiting conditions and on Pi deficiency-induced accumulation of amino acids and organic acids, which play important roles in Pi use efficiency. Fe deficiency in Pi replete conditions hardly changed growth and metabolite profiles in roots and shoots of Arabidopsis thaliana, but partially rescued growth under conditions of Pi starvation and severely modulated Pi deficiency-induced metabolic adjustments. Analysis of T-DNA insertion lines revealed the concerted coordination of metabolic profiles by regulators of Fe (FIT, bHLH104, BRUTUS, PYE) as well as of Pi (SPX1, PHR1, PHL1, bHLH32) starvation responses. The results show the interdependency of Pi and Fe availability and the interplay between Pi and Fe starvation signaling on the generation of plant metabolite profiles. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 482 TI - Root responses to aluminium and iron stresses require the SIZ1 SUMO ligase to modulate the STOP1 transcription factor JO - Plant J. PY - 2021 SP - 1507-1521 AU - Mercier, C. AU - Roux, B. AU - Havé, M. AU - Le Poder, L. AU - Duong, N. AU - David, P. AU - Leonhardt, N. AU - Blanchard, L. AU - Naumann, C. AU - Abel, S. AU - Cuyas, L. AU - Pluchon, S. AU - Laurent, N. AU - Desnos, T. AU - VL - 108 UR - https://doi.org/10.1111/tpj.15525 DO - 10.1111/tpj.15525 AB - STOP1, an Arabidopsis transcription factor favouring root growth tolerance against Al toxicity, acts in the response to iron under low Pi (-Pi). Previous studies have shown that Al and Fe regulate the stability and accumulation of STOP1 in roots, and that the STOP1 protein is sumoylated by an unknown E3 ligase. Here, using a forward genetics suppressor screen, we identified the E3 SUMO (small ubiquitin-like modifier) ligase SIZ1 as a modulator of STOP1 signalling. Mutations in SIZ1 increase the expression of ALMT1 (a direct target of STOP1) and root growth responses to Al and Fe stress in a STOP1-dependent manner. Moreover, loss-of-function mutations in SIZ1 enhance the abundance of STOP1 in the root tip. However, no sumoylated STOP1 protein was detected by western blot analysis in our sumoylation assay in E. coli, suggesting the presence of a more sophisticated mechanism. We conclude that the sumo ligase SIZ1 negatively regulates STOP1 signalling, at least in part by modulating STOP1 protein in the root tip. Our results will allow a better understanding of this signalling pathway. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 469 TI - IQ67 DOMAIN proteins facilitate preprophase band formation and division-plane orientation JO - Nat. Plants PY - 2021 SP - 739-747 AU - Kumari, P. AU - Dahiya, P. AU - Livanos, P. AU - Zergiebel, L. AU - Kölling, M. AU - Poeschl, Y. AU - Stamm, G. AU - Hermann, A. AU - Abel, S. AU - Müller, S. AU - Bürstenbinder, K. AU - VL - 7 UR - https://doi.org/10.1038/s41477-021-00923-z DO - 10.1038/s41477-021-00923-z AB - Spatiotemporal control of cell division is essential for the growth and development of multicellular organisms. In plant cells, proper cell plate insertion during cytokinesis relies on the premitotic establishment of the division plane at the cell cortex. Two plant-specific cytoskeleton arrays, the preprophase band (PPB) and the phragmoplast, play important roles in division-plane orientation and cell plate formation, respectively1. Microtubule organization and dynamics and their communication with membranes at the cortex and cell plate are coordinated by multiple, mostly distinct microtubule-associated proteins2. How division-plane selection and establishment are linked, however, is still unknown. Here, we report members of the Arabidopsis IQ67 DOMAIN (IQD) family3 as microtubule-targeted proteins that localize to the PPB and phragmoplast and additionally reside at the cell plate and a polarized cortical region including the cortical division zone (CDZ). IQDs physically interact with PHRAGMOPLAST ORIENTING KINESIN (POK) proteins4,5 and PLECKSTRIN HOMOLOGY GTPase ACTIVATING (PHGAP) proteins6, which are core components of the CDZ1. The loss of IQD function impairs PPB formation and affects CDZ recruitment of POKs and PHGAPs, resulting in division-plane positioning defects. We propose that IQDs act as cellular scaffolds that facilitate PPB formation and CDZ set-up during symmetric cell division. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2520 TI - A cross-kingdom conserved ER-phagy receptor maintains endoplasmic reticulum homeostasis during stress JO - bioRxiv PY - 2020 SP - AU - Stephani, M. AU - Picchianti, L. AU - Gajic, A. AU - Beveridge, R. AU - Skarwan, E. AU - Sanchez de Medina Hernandez, V. AU - Mohseni, A. AU - Clavel, M. AU - Zeng, Y. AU - Naumann, C. AU - Matuszkiewicz, M. AU - Turco, E. AU - Loefke, C. AU - Li, B. AU - Durnberger, G. AU - Schutzbier, M. AU - Chen, H. T. AU - Abdrakhmanov, A. AU - Savova, A. AU - Chia, K.-S. AU - Djamei, A. AU - Schaffner, I. AU - Abel, S. AU - Jiang, L. AU - Mechtler, K. AU - Ikeda, F. AU - Martens, S. AU - Clausen, T. AU - Dagdas, Y. AU - VL - UR - DO - 10.1101/2020.03.18.995316 AB - Eukaryotes have evolved various quality control mechanisms to promote proteostasis in the ER. Selective removal of certain ER domains via autophagy (termed as ER-phagy) has emerged as a major quality control mechanism. However, the degree to which ER-phagy is employed by other branches of ER-quality control remains largely elusive. Here, we identify a cytosolic protein, C53, that is specifically recruited to autophagosomes during ER-stress, in both plant and mammalian cells. C53 interacts with ATG8 via a distinct binding epitope, featuring a shuffled ATG8 interacting motif (sAIM). C53 senses proteotoxic stress in the ER lumen by forming a tripartite receptor complex with the ER-associated ufmylation ligase UFL1 and its membrane adaptor DDRGK1. The C53/UFL1/DDRGK1 receptor complex is activated by stalled ribosomes and induces the degradation of internal or passenger proteins in the ER. Consistently, the C53 receptor complex and ufmylation mutants are highly susceptible to ER stress. Thus, C53 forms an ancient quality control pathway that bridges selective autophagy with ribosome-associated quality control at the ER. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2517 TI - Reshaping of the Arabidopsis thaliana proteome landscape and co-regulation of proteins in development and immunity JO - bioRxiv PY - 2020 SP - AU - Bassal, M. AU - Majovsky, P. AU - Thieme, D. AU - Herr, T. AU - Abukhalaf, M. AU - Ayash, M. AU - Al Shweiki, M. R. AU - Proksch, C. AU - Hmedat, A. AU - Ziegler, J. AU - Neumann, S. AU - Hoehenwarter, W. AU - VL - UR - https://doi.org/10.1101/2020.03.09.978627 DO - 10.1101/2020.03.09.978627 AB - Proteome remodeling is a fundamental adaptive response and proteins in complex and functionally related proteins are often co-expressed. Using a deep sampling strategy we define Arabidopsis thaliana tissue core proteomes at around 10,000 proteins per tissue and absolutely quantify (copy numbers per cell) nearly 16,000 proteins throughout the plant lifecycle. A proteome wide survey of global post translational modification revealed amino acid exchanges pointing to potential conservation of translational infidelity in eukaryotes. Correlation analysis of protein abundance uncovered potentially new tissue and age specific roles of entire signaling modules regulating transcription in photosynthesis, seed development and senescence and abscission. Among others, the data suggest a potential function of RD26 and other NAC transcription factors in seed development related to desiccation tolerance as well as a possible function of Cysteine-rich Receptor-like Kinases (CRKs) as ROS sensors in senescence. All of the components of ribosome biogenesis factor (RBF) complexes were co-expressed tissue and age specifically indicating functional promiscuity in the assembly of these little described protein complexes in Arabidopsis. Treatment of seedlings with flg22 for 16 hours allowed us to characterize proteome architecture in basal immunity in detail. The results were complemented with parallel reaction monitoring (PRM) targeted proteomics, phytohormone, amino acid and transcript measurements. We obtained strong evidence of suppression of jasmonate (JA) and JA-Ile levels by deconjugation and hydroxylation via IAA-ALA RESISTANT3 (IAR3) and JASMONATE-INDUCED OXYGENASE 2 (JOX2) under the control of JASMONATE INSENSITIVE 1 (MYC2). This previously unknown regulatory switch is another part of the puzzle of the as yet understudied role of JA in pattern triggered immunity. The extensive coverage of the Arabidopsis proteome in various biological scenarios presents a rich resource to plant biologists that we make available to the community. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 536 TI - Reshaping of the Arabidopsis thaliana Proteome Landscape and Co-regulation of Proteins in Development and Immunity JO - Mol. Plant PY - 2020 SP - 1709-1732 AU - Bassal, M. AU - Abukhalaf, M. AU - Majovsky, P. AU - Thieme, D. AU - Herr, T. AU - Ayash, M. AU - Tabassum, N. AU - Al Shweiki, M. R. AU - Proksch, C. AU - Hmedat, A. AU - Ziegler, J. AU - Lee, J. AU - Neumann, S. AU - Hoehenwarter, W. AU - VL - 13 UR - DO - 10.1016/j.molp.2020.09.024 AB - Proteome remodeling is a fundamental adaptive response, and proteins in complexes and functionally related proteins are often co-expressed. Using a deep sampling strategy we define core proteomes of Arabidopsis thaliana tissues with around 10 000 proteins per tissue, and absolutely quantify (copy numbers per cell) nearly 16 000 proteins throughout the plant lifecycle. A proteome-wide survey of global post-translational modification revealed amino acid exchanges pointing to potential conservation of translational infidelity in eukaryotes. Correlation analysis of protein abundance uncovered potentially new tissue- and age-specific roles of entire signaling modules regulating transcription in photosynthesis, seed development, and senescence and abscission. Among others, the data suggest a potential function of RD26 and other NAC transcription factors in seed development related to desiccation tolerance as well as a possible function of cysteine-rich receptor-like kinases (CRKs) as ROS sensors in senescence. All of the components of ribosome biogenesis factor (RBF) complexes were found to be co-expressed in a tissue- and age-specific manner, indicating functional promiscuity in the assembly of these less-studied protein complexes in Arabidopsis. Furthermore, we characterized detailed proteome remodeling in basal immunity by treating Arabidopsis seeldings with flg22. Through simultaneously monitoring phytohormone and transcript changes upon flg22 treatment, we obtained strong evidence of suppression of jasmonate (JA) and JA-isoleucine (JA-Ile) levels by deconjugation and hydroxylation by IAA-ALA RESISTANT3 (IAR3) and JASMONATE-INDUCED OXYGENASE 2 (JOX2), respectively, under the control of JASMONATE INSENSITIVE 1 (MYC2), suggesting an unrecognized role of a new JA regulatory switch in pattern-triggered immunity. Taken together, the datasets generated in this study present extensive coverage of the Arabidopsis proteome in various biological scenarios, providing a rich resource available to the whole plant science community. A2 - C1 - Biochemistry of Plant Interactions; Molecular Signal Processing ER - TY - JOUR ID - 606 TI - A cross-kingdom conserved ER-phagy receptor maintains endoplasmic reticulum homeostasis during stress JO - eLife PY - 2020 SP - e58396 AU - Stephani, M. AU - Picchianti, L. AU - Gajic, A. AU - Beveridge, R. AU - Skarwan, E. AU - Sanchez de Medina Hernandez, V. AU - Mohseni, A. AU - Clavel, M. AU - Zeng, Y. AU - Naumann, C. AU - Matuszkiewicz, M. AU - Turco, E. AU - Loefke, C. AU - Li, B. AU - Durnberger, G. AU - Schutzbier, M. AU - Chen, H. T. AU - Abdrakhmanov, A. AU - Savova, A. AU - Chia, K.-S. AU - Djamei, A. AU - Schaffner, I. AU - Abel, S. AU - Jiang, L. AU - Mechtler, K. AU - Ikeda, F. AU - Martens, S. AU - Clausen, T. AU - Dagdas, Y. AU - VL - 9 UR - https://elifesciences.org/articles/58396 DO - 10.7554/elife.58396 AB - Eukaryotes have evolved various quality control mechanisms to promote proteostasis in the endoplasmic reticulum (ER). Selective removal of certain ER domains via autophagy (termed as ER-phagy) has emerged as a major quality control mechanism. However, the degree to which ER-phagy is employed by other branches of ER-quality control remains largely elusive. Here, we identify a cytosolic protein, C53, that is specifically recruited to autophagosomes during ER-stress, in both plant and mammalian cells. C53 interacts with ATG8 via a distinct binding epitope, featuring a shuffled ATG8 interacting motif (sAIM). C53 senses proteotoxic stress in the ER lumen by forming a tripartite receptor complex with the ER-associated ufmylation ligase UFL1 and its membrane adaptor DDRGK1. The C53/UFL1/DDRGK1 receptor complex is activated by stalled ribosomes and induces the degradation of internal or passenger proteins in the ER. Consistently, the C53 receptor complex and ufmylation mutants are highly susceptible to ER stress. Thus, C53 forms an ancient quality control pathway that bridges selective autophagy with ribosome-associated quality control in the ER. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 745 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 - 722 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 - 640 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 - 635 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 - 633 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 - 684 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 - 683 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 - 702 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 - 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 - JOUR ID - 760 TI - Predictive value of hypoglycin A and methylencyclopropylacetic acid conjugates in a horse with atypical myopathy in comparison to its cograzing partners JO - Equine Vet. Educ. PY - 2018 SP - 24-28 AU - Bochnia, M. AU - Scheidemann, W. AU - Ziegler, J. AU - Sander, J. AU - Vollstedt, S. AU - Glatter, M. AU - Janzen, N. AU - Terhardt, M. AU - Zeyner, A. AU - VL - 30 UR - DO - 10.1111/eve.12596 AB - Hypoglycin A (HGA) was detected in blood and urine of a horse suffering from atypical myopathy (AM; Day 2, serum, 8290 μg/l; urine: Day 1, 574, Day 2, 742 μg/l) and in its cograzing partners with a high variability (46–1570 μg/l serum). Over the period of disease, the level of the toxic metabolites (methylencyclopropylacetic acid [MCPA]‐conjugates) increased in body fluids of the AM horse (MCPA‐carnitine: Day 2, 0.246, Day 3, 0.581 μmol/l serum; MCPA‐carnitine: Day 2, 0.621, Day 3, 0.884 μmol/mmol creatinine in urine) and HGA decreased rapidly (Day 3, 2430 μg/l serum). In cograzing horses MCPA‐conjugates were not detected. HGA in seeds ranged from 268 to 367 μg/g. Although HGA was present in body fluids of healthy cograzing horses, MCPA‐conjugates were not detectable, in contrast to the AM horse. Therefore, increasing concentrations of MCPA‐conjugates are supposed to be linked with the onset of AM and both parameters seem to indicate the clinical stage of disease. However, detection of HGA in body fluids of cograzing horses might be a promising step in preventing the disease. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 798 TI - Identification of hypoglycin A binding adsorbents as potential preventive measures in co-grazers of atypical myopathy affected horses JO - Equine Vet. J. PY - 2018 SP - 220-227 AU - Krägeloh, T. AU - Cavalleri, J. M. V. AU - Ziegler, J. AU - Sander, J. AU - Terhardt, M. AU - Breves, G. AU - Cehak, A. AU - VL - 50 UR - DO - 10.1111/evj.12723 AB - BackgroundIntestinal absorption of hypoglycin A (HGA) and its metabolism are considered major prerequisites for atypical myopathy (AM). The increasing incidence and the high mortality rate of AM urgently necessitate new therapeutic and/or preventative approaches.ObjectivesTo identify a substance for oral administration capable of binding HGA in the intestinal lumen and effectively reducing the intestinal absorption of the toxin.Study designExperimental in vitro study.MethodsSubstances commonly used in equine practice (activated charcoal composition, di‐tri‐octahedral smectite, mineral oil and activated charcoal) were tested for their binding capacity for HGA using an in vitro incubation method. The substance most effective in binding HGA was subsequently tested for its potential to reduce intestinal HGA absorption. Jejunal tissues of 6 horses were incubated in Ussing chambers to determine mucosal uptake, tissue accumulation, and serosal release of HGA in the presence and absence of the target substance. Potential intestinal metabolism in methylenecyclopropyl acetic acid (MCPA)‐conjugates was investigated by analysing their concentrations in samples from the Ussing chambers.ResultsActivated charcoal composition and activated charcoal were identified as potent HGA binding substances with dose and pH dependent binding capacity. There was no evidence of intestinal HGA metabolism.Main limitationsBinding capacity of adsorbents was tested in vitro using aqueous solutions, and in vivo factors such as transit time and composition of intestinal content, may affect adsorption capacity after oral administration.ConclusionsFor the first time, this study identifies substances capable of reducing HGA intestinal absorption. This might have major implications as a preventive measure in cograzers of AM affected horses but also in horses at an early stage of intoxication. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 795 TI - Inhibition of phospholipases influences the metabolism of wound-induced benzylisoquinoline alkaloids in Papaver somniferum L. JO - J. Plant Physiol. PY - 2018 SP - 1-8 AU - Jablonická, V. AU - Ziegler, J. AU - Vatehová, Z. AU - Lišková, D. AU - Heilmann, I. AU - Obložinský, M. AU - Heilmann, M. AU - VL - 223 UR - DO - 10.1016/j.jplph.2018.01.007 AB - Benzylisoquinoline alkaloids (BIAs) are important secondary plant metabolites and include medicinally relevant drugs, such as morphine or codeine. As the de novo synthesis of BIA backbones is (still) unfeasible, to date the opium poppy plant Papaver somniferum L. represents the main source of BIAs. The formation of BIAs is induced in poppy plants by stress conditions, such as wounding or salt treatment; however, the details about regulatory processes controlling BIA formation in opium poppy are not well studied. Environmental stresses, such as wounding or salinization, are transduced in plants by phospholipid-based signaling pathways, which involve different classes of phospholipases. Here we investigate whether pharmacological inhibition of phospholipase A2 (PLA2, inhibited by aristolochic acid (AA)) or phospholipase D (PLD; inhibited by 5-fluoro-2-indolyl des-chlorohalopemide (FIPI)) in poppy plants influences wound-induced BIA accumulation and the expression of key biosynthetic genes. We show that inhibition of PLA2 results in increased morphinan biosynthesis concomitant with reduced production of BIAs of the papaverine branch, whereas inhibition of PLD results in increased production of BIAs of the noscapine branch. The data suggest that phospholipid-dependent signaling pathways contribute to the activation of morphine biosynthesis at the expense of the production of other BIAs in poppy plants. A better understanding of the effectors and the principles of regulation of alkaloid biosynthesis might be the basis for the future genetic modification of opium poppy to optimize BIA production. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 960 TI - Arabidopsis Transporter ABCG37/PDR9 contributes primarily highly oxygenated Coumarins to Root Exudation JO - Sci. Rep. PY - 2017 SP - 3704 AU - Ziegler, J. AU - Schmidt, S. AU - Strehmel, N. AU - Scheel, D. AU - Abel, S. AU - VL - 7 UR - DO - 10.1038/s41598-017-03250-6 AB - The chemical composition of root exudates strongly impacts the interactions of plants with microorganisms in the rhizosphere and the efficiency of nutrient acquisition. Exudation of metabolites is in part mediated by ATP-binding cassette (ABC) transporters. In order to assess the contribution of individual ABC transporters to root exudation, we performed an LC-MS based non-targeted metabolite profiling of semi-polar metabolites accumulating in root exudates of Arabidopsis thaliana plants and mutants deficient in the expression of ABCG36 (PDR8/PEN3), ABCG37 (PDR9) or both transporters. Comparison of the metabolite profiles indicated distinct roles for each ABC transporter in root exudation. Thymidine exudation could be attributed to ABCG36 function, whereas coumarin exudation was strongly reduced only in ABCG37 deficient plants. However, coumarin exudation was compromised in abcg37 mutants only with respect to certain metabolites of this substance class. The specificity of ABCG37 for individual coumarins was further verified by a targeted LC-MS based coumarin profiling method. The response to iron deficiency, which is known to strongly induce coumarin exudation, was also investigated. In either treatment, the distribution of individual coumarins between roots and exudates in the investigated genotypes suggested the involvement of ABCG37 in the exudation specifically of highly oxygenated rather than monohydroxylated coumarins. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 862 TI - Low phosphate activates STOP1-ALMT1 to rapidly inhibit root cell elongation JO - Nat. Commun. PY - 2017 SP - 15300 AU - Balzergue, C. AU - Dartevelle, T. AU - Godon, C. AU - Laugier, E. AU - Meisrimler, C. AU - Teulon, J.-M. AU - Creff, A. AU - Bissler, M. AU - Brouchoud, C. AU - Hagège, A. AU - Müller, J. AU - Chiarenza, S. AU - Javot, H. AU - Becuwe-Linka, N. AU - David, P. AU - Péret, B. AU - Delannoy, E. AU - Thibaud, M.-C. AU - Armengaud, J. AU - Abel, S. AU - Pellequer, J.-L. AU - Nussaume, L. AU - Desnos, T. AU - VL - 8 UR - DO - 10.1038/ncomms15300 AB - Environmental cues profoundly modulate cell proliferation and cell elongation to inform and direct plant growth and development. External phosphate (Pi) limitation inhibits primary root growth in many plant species. However, the underlying Pi sensory mechanisms are unknown. Here we genetically uncouple two Pi sensing pathways in the root apex of Arabidopsis thaliana. First, the rapid inhibition of cell elongation in the transition zone is controlled by transcription factor STOP1, by its direct target, ALMT1, encoding a malate channel, and by ferroxidase LPR1, which together mediate Fe and peroxidase-dependent cell wall stiffening. Second, during the subsequent slow inhibition of cell proliferation in the apical meristem, which is mediated by LPR1-dependent, but largely STOP1–ALMT1-independent, Fe and callose accumulate in the stem cell niche, leading to meristem reduction. Our work uncovers STOP1 and ALMT1 as a signalling pathway of low Pi availability and exuded malate as an unexpected apoplastic inhibitor of root cell wall expansion. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 855 TI - Phosphate scouting by root tips JO - Curr. Opin. Plant Biol. PY - 2017 SP - 168-177 AU - Abel, S. AU - VL - 39 UR - DO - 10.1016/j.pbi.2017.04.016 AB - Chemistry assigns phosphate (Pi) dominant roles in metabolism; however, it also renders the macronutrient a genuinely limiting factor of plant productivity. Pi bioavailability is restricted by low Pi mobility in soil and antagonized by metallic toxicities, which force roots to actively seek and selectively acquire the vital element. During the past few years, a first conceptual outline has emerged of the sensory mechanisms at root tips, which monitor external Pi and transmit the edaphic cue to inform root development. This review highlights new aspects of the Pi acquisition strategy of Arabidopsis roots, as well as a framework of local Pi sensing in the context of antagonistic interactions between Pi and its major associated metallic cations, Fe3+ and Al3+. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 906 TI - Botrytis cinerea B05.10 promotes disease development in Arabidopsis by suppressing WRKY33-mediated host immunity JO - Plant Cell Environ. PY - 2017 SP - 2189-2206 AU - Liu, S. AU - Ziegler, J. AU - Zeier, J. AU - Birkenbihl, R. P. AU - Somssich, I. E. AU - VL - 40 UR - DO - 10.1111/pce.13022 AB - The large WRKY transcription factor family is mainly involved in regulating plant immune responses. Arabidopsis WRKY33 is a key transcriptional regulator of hormonal and metabolic processes towards Botrytis cinerea strain 2100 infection and is essential for resistance. In contrast to B. cinerea strain 2100, the strain B05.10 is virulent on wild‐type (WT) Col‐0 Arabidopsis plants highlighting the genetic diversity within this pathogen species. We analysed how early WRKY33‐dependent responses are affected upon infection with strain B05.10 and found that most of these responses were strongly dampened during this interaction. Ectopic expression of WRKY33 resulted in complete resistance towards this strain indicating that virulence of B05.10, at least partly, depends on suppressing WRKY33 expression/protein accumulation. As a consequence, the expression levels of direct WRKY33 target genes, including those involved in the biosynthesis of camalexin, were also reduced upon infection. Concomitantly, elevated levels of the phytohormone abscisic acid (ABA) were observed. Molecular and genetic studies revealed that ABA negatively influences defence to B05.10 and effects jasmonic acid/ethylene (JA/ET) and salicylic acid (SA) levels. Susceptibility/resistance was determined by the antagonistic effect of ABA on JA, and this crosstalk required suppressing WRKY33 functions at early infection stages. This indicates that B. cinerea B05.10 promotes disease by suppressing WRKY33‐mediated host defences. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1051 TI - Piriformospora indica Stimulates Root Metabolism of Arabidopsis thaliana JO - Int. J. Mol. Sci. PY - 2016 SP - 1091 AU - Strehmel, N. AU - Mönchgesang, S. AU - Herklotz, S. AU - Krüger, S. AU - Ziegler, J. AU - Scheel, D. AU - VL - 17 UR - DO - 10.3390/ijms17071091 AB - Piriformospora indica is a root-colonizing fungus, which interacts with a variety of plants including Arabidopsis thaliana. This interaction has been considered as mutualistic leading to growth promotion of the host. So far, only indolic glucosinolates and phytohormones have been identified as key players. In a comprehensive non-targeted metabolite profiling study, we analyzed Arabidopsis thaliana’s roots, root exudates, and leaves of inoculated and non-inoculated plants by ultra performance liquid chromatography/electrospray ionization quadrupole-time-of-flight mass spectrometry (UPLC/(ESI)-QTOFMS) and gas chromatography/electron ionization quadrupole mass spectrometry (GC/EI-QMS), and identified further biomarkers. Among them, the concentration of nucleosides, dipeptides, oligolignols, and glucosinolate degradation products was affected in the exudates. In the root profiles, nearly all metabolite levels increased upon co-cultivation, like carbohydrates, organic acids, amino acids, glucosinolates, oligolignols, and flavonoids. In the leaf profiles, we detected by far less significant changes. We only observed an increased concentration of organic acids, carbohydrates, ascorbate, glucosinolates and hydroxycinnamic acids, and a decreased concentration of nitrogen-rich amino acids in inoculated plants. These findings contribute to the understanding of symbiotic interactions between plant roots and fungi of the order of Sebacinales and are a valid source for follow-up mechanistic studies, because these symbioses are particular and clearly different from interactions of roots with mycorrhizal fungi or dark septate endophytes A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1004 TI - Comparative expression profiling reveals a role of the root apoplast in local phosphate response JO - BMC Plant Biol. PY - 2016 SP - 106 AU - Hoehenwarter, W. AU - Mönchgesang, S. AU - Neumann, S. AU - Majovsky, P. AU - Abel, S. AU - Müller, J. AU - VL - 16 UR - DO - 10.1186/s12870-016-0790-8 AB - BackgroundPlant adaptation to limited phosphate availability comprises a wide range of responses to conserve and remobilize internal phosphate sources and to enhance phosphate acquisition. Vigorous restructuring of root system architecture provides a developmental strategy for topsoil exploration and phosphate scavenging. Changes in external phosphate availability are locally sensed at root tips and adjust root growth by modulating cell expansion and cell division. The functionally interacting Arabidopsis genes, LOW PHOSPHATE RESPONSE 1 and 2 (LPR1/LPR2) and PHOSPHATE DEFICIENCY RESPONSE 2 (PDR2), are key components of root phosphate sensing. We recently demonstrated that the LOW PHOSPHATE RESPONSE 1 - PHOSPHATE DEFICIENCY RESPONSE 2 (LPR1-PDR2) module mediates apoplastic deposition of ferric iron (Fe3+) in the growing root tip during phosphate limitation. Iron deposition coincides with sites of reactive oxygen species generation and triggers cell wall thickening and callose accumulation, which interfere with cell-to-cell communication and inhibit root growth.ResultsWe took advantage of the opposite phosphate-conditional root phenotype of the phosphate deficiency response 2 mutant (hypersensitive) and low phosphate response 1 and 2 double mutant (insensitive) to investigate the phosphate dependent regulation of gene and protein expression in roots using genome-wide transcriptome and proteome analysis. We observed an overrepresentation of genes and proteins that are involved in the regulation of iron homeostasis, cell wall remodeling and reactive oxygen species formation, and we highlight a number of candidate genes with a potential function in root adaptation to limited phosphate availability. Our experiments reveal that FERRIC REDUCTASE DEFECTIVE 3 mediated, apoplastic iron redistribution, but not intracellular iron uptake and iron storage, triggers phosphate-dependent root growth modulation. We further highlight expressional changes of several cell wall-modifying enzymes and provide evidence for adjustment of the pectin network at sites of iron accumulation in the root.ConclusionOur study reveals new aspects of the elaborate interplay between phosphate starvation responses and changes in iron homeostasis. The results emphasize the importance of apoplastic iron redistribution to mediate phosphate-dependent root growth adjustment and suggest an important role for citrate in phosphate-dependent apoplastic iron transport. We further demonstrate that root growth modulation correlates with an altered expression of cell wall modifying enzymes and changes in the pectin network of the phosphate-deprived root tip, supporting the hypothesis that pectins are involved in iron binding and/or phosphate mobilization. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 979 TI - Structural Biology of Nuclear Auxin Action JO - Trends Plant Sci. PY - 2016 SP - 302-316 AU - Dinesh, D. C. AU - Calderón Villalobos, L. I. A. AU - Abel, S. AU - VL - 21 UR - DO - 10.1016/j.tplants.2015.10.019 AB - Auxin coordinates plant development largely via hierarchical control of gene expression. During the past decades, the study of early auxin genes paired with the power of Arabidopsis genetics have unraveled key nuclear components and molecular interactions that perceive the hormone and activate primary response genes. Recent research in the realm of structural biology allowed unprecedented insight into: (i) the recognition of auxin-responsive DNA elements by auxin transcription factors; (ii) the inactivation of those auxin response factors by early auxin-inducible repressors; and (iii) the activation of target genes by auxin-triggered repressor degradation. The biophysical studies reviewed here provide an impetus for elucidating the molecular determinants of the intricate interactions between core components of the nuclear auxin response module. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1069 TI - Non-targeted profiling of semi-polar metabolites in Arabidopsis root exudates uncovers a role for coumarin secretion and lignification during the local response to phosphate limitation JO - J. Exp. Bot. PY - 2016 SP - 1421-1432 AU - Ziegler, J. AU - Schmidt, S. AU - Chutia, R. AU - Müller, J. AU - Böttcher, C. AU - Strehmel, N. AU - Scheel, D. AU - Abel, S. AU - VL - 67 UR - DO - 10.1093/jxb/erv539 AB - Plants have evolved two major strategies to cope with phosphate (Pi) limitation. The systemic response, mainly comprising increased Pi uptake and metabolic adjustments for more efficient Pi use, and the local response, enabling plants to explore Pi-rich soil patches by reorganization of the root system architecture. Unlike previous reports, this study focused on root exudation controlled by the local response to Pi deficiency. To approach this, a hydroponic system separating the local and systemic responses was developed. Arabidopsis thaliana genotypes exhibiting distinct sensitivities to Pi deficiency could be clearly distinguished by their root exudate composition as determined by non-targeted reversed-phase ultraperformance liquid chromatography electrospray ionization quadrupole-time-of-flight mass spectrometry metabolite profiling. Compared with wild-type plants or insensitive low phosphate root 1 and 2 (lpr1 lpr2) double mutant plants, the hypersensitive phosphate deficiency response 2 (pdr2) mutant exhibited a reduced number of differential features in root exudates after Pi starvation, suggesting the involvement of PDR2-encoded P5-type ATPase in root exudation. Identification and analysis of coumarins revealed common and antagonistic regulatory pathways between Pi and Fe deficiency-induced coumarin secretion. The accumulation of oligolignols in root exudates after Pi deficiency was inversely correlated with Pi starvation-induced lignification at the root tips. The strongest oligolignol accumulation in root exudates was observed for the insensitive lpr1 lpr2 double mutant, which was accompanied by the absence of Pi deficiency-induced lignin deposition, suggesting a role of LPR ferroxidases in lignin polymerization during Pi starvation. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1087 TI - Solution structure of the PsIAA4 oligomerization domain reveals interaction modes for transcription factors in early auxin response JO - Proc. Natl. Acad. Sci. U.S.A. PY - 2015 SP - 6230-6235 AU - Dinesh, D. C. AU - Kovermann, M. AU - Gopalswamy, M. AU - Hellmuth, A. AU - Calderón Villalobos, L. I. A. AU - Lilie, H. AU - Balbach, J. AU - Abel, S. AU - VL - 112 UR - DO - 10.1073/pnas.1424077112 AB - The plant hormone auxin activates primary response genes by facilitating proteolytic removal of AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA)-inducible repressors, which directly bind to transcriptional AUXIN RESPONSE FACTORS (ARF). Most AUX/IAA and ARF proteins share highly conserved C-termini mediating homotypic and heterotypic interactions within and between both protein families. The high-resolution NMR structure of C-terminal domains III and IV of the AUX/IAA protein PsIAA4 from pea (Pisum sativum) revealed a globular ubiquitin-like β-grasp fold with homologies to the Phox and Bem1p (PB1) domain. The PB1 domain of wild-type PsIAA4 features two distinct surface patches of oppositely charged amino acid residues, mediating front-to-back multimerization via electrostatic interactions. Mutations of conserved basic or acidic residues on either face suppressed PsIAA4 PB1 homo-oligomerization in vitro and confirmed directional interaction of full-length PsIAA4 in vivo (yeast two-hybrid system). Mixing of oppositely mutated PsIAA4 PB1 monomers enabled NMR mapping of the negatively charged interface of the reconstituted PsIAA4 PB1 homodimer variant, whose stoichiometry (1:1) and equilibrium binding constant (KD ∼6.4 μM) were determined by isothermal titration calorimetry. In silico protein–protein docking studies based on NMR and yeast interaction data derived a model of the PsIAA4 PB1 homodimer, which is comparable with other PB1 domain dimers, but indicated considerable differences between the homodimeric interfaces of AUX/IAA and ARF PB1 domains. Our study provides an impetus for elucidating the molecular determinants that confer specificity to complex protein–protein interaction circuits between members of the two central families of transcription factors important to the regulation of auxin-responsive gene expression. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1084 TI - Perturbations in the Primary Metabolism of Tomato and Arabidopsis thaliana Plants Infected with the Soil-Borne Fungus Verticillium dahliae JO - PLOS ONE PY - 2015 SP - e0138242 AU - Buhtz, A. AU - Witzel, K. AU - Strehmel, N. AU - Ziegler, J. AU - Abel, S. AU - Grosch, R. AU - VL - 10 UR - DO - 10.1371/journal.pone.0138242 AB - The hemibiotrophic soil-borne fungus Verticillium dahliae is a major pathogen of a number of economically important crop species. Here, the metabolic response of both tomato and Arabidopsis thaliana to V. dahliae infection was analysed by first using non-targeted GC-MS profiling. The leaf content of both major cell wall components glucuronic acid and xylose was reduced in the presence of the pathogen in tomato but enhanced in A. thaliana. The leaf content of the two tricarboxylic acid cycle intermediates fumaric acid and succinic acid was increased in the leaf of both species, reflecting a likely higher demand for reducing equivalents required for defence responses. A prominent group of affected compounds was amino acids and based on the targeted analysis in the root, it was shown that the level of 12 and four free amino acids was enhanced by the infection in, respectively, tomato and A. thaliana, with leucine and histidine being represented in both host species. The leaf content of six free amino acids was reduced in the leaf tissue of diseased A. thaliana plants, while that of two free amino acids was raised in the tomato plants. This study emphasizes the role of primary plant metabolites in adaptive responses when the fungus has colonized the plant. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1077 TI - Hypoglycin A Content in Blood and Urine Discriminates Horses with Atypical Myopathy from Clinically Normal Horses Grazing on the Same Pasture JO - PLOS ONE PY - 2015 SP - e0136785 AU - Bochnia, M. AU - Ziegler, J. AU - Sander, J. AU - Uhlig, A. AU - Schaefer, S. AU - Vollstedt, S. AU - Glatter, M. AU - Abel, S. AU - Recknagel, S. AU - Schusser, G. F. AU - Wensch-Dorendorf, M. AU - Zeyner, A. AU - VL - 10 UR - DO - 10.1371/journal.pone.0136785 AB - Hypoglycin A (HGA) in seeds of Acer spp. is suspected to cause seasonal pasture myopathy in North America and equine atypical myopathy (AM) in Europe, fatal diseases in horses on pasture. In previous studies, this suspicion was substantiated by the correlation of seed HGA content with the concentrations of toxic metabolites in urine and serum (MCPA-conjugates) of affected horses. However, seed sampling was conducted after rather than during an outbreak of the disease. The aim of this study was to further confirm the causality between HGA occurrence and disease outbreak by seed sampling during an outbreak and the determination of i) HGA in seeds and of ii) HGA and MCPA-conjugates in urine and serum of diseased horses. Furthermore, cograzing healthy horses, which were present on AM affected pastures, were also investigated. AM-pastures in Germany were visited to identify seeds of Acer pseudoplatanus and serum (n = 8) as well as urine (n = 6) from a total of 16 diseased horses were analyzed for amino acid composition by LC-ESI-MS/MS, with a special focus on the content of HGA. Additionally, the content of its toxic metabolite was measured in its conjugated form in body fluids (UPLC-MS/MS). The seeds contained 1.7–319.8 μg HGA/g seed. The content of HGA in serum of affected horses ranged from 387.8–8493.8 μg/L (controls < 10 μg/L), and in urine from 143.8–926.4 μg/L (controls < 10 μg/L), respectively. Healthy cograzing horses on AM-pastures showed higher serum (108.8 ± 83.76 μg/L) and urine concentrations (26.9 ± 7.39 μg/L) compared to control horses, but lower concentrations compared to diseased horses. The range of MCPA-carnitine and creatinine concentrations found in diseased horses in serum and urine were 0.17–0.65 mmol/L (controls < 0.01), and 0.34–2.05 μmol/mmoL (controls < 0.001), respectively. MCPA-glycine levels in urine of cograzing horses were higher compared to controls. Thus, the causal link between HGA intoxication and disease outbreak could be further substantiated, and the early detection of HGA in cograzing horses, which are clinically normal, might be a promising step in prophylaxis. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1136 TI - Iron-Dependent Callose Deposition Adjusts Root Meristem Maintenance to Phosphate Availability JO - Dev. Cell PY - 2015 SP - 216-230 AU - Müller, J. AU - Toev, T. AU - Heisters, M. AU - Teller, J. AU - Moore, K. AU - Hause, G. AU - Dinesh, D. AU - Bürstenbinder, K. AU - Abel, S. AU - VL - 33 UR - DO - 10.1016/j.devcel.2015.02.007 AB - Plant root development is informed by numerous edaphic cues. Phosphate (Pi) availability impacts the root system architecture by adjusting meristem activity. However, the sensory mechanisms monitoring external Pi status are elusive. Two functionally interacting Arabidopsis genes, LPR1 (ferroxidase) and PDR2 (P5-type ATPase), are key players in root Pi sensing, which is modified by iron (Fe) availability. We show that the LPR1-PDR2 module facilitates, upon Pi limitation, cell-specific apoplastic Fe and callose deposition in the meristem and elongation zone of primary roots. Expression of cell-wall-targeted LPR1 determines the sites of Fe accumulation as well as callose production, which interferes with symplastic communication in the stem cell niche, as demonstrated by impaired SHORT-ROOT movement. Antagonistic interactions of Pi and Fe availability control primary root growth via meristem-specific callose formation, likely triggered by LPR1-dependent redox signaling. Our results link callose-regulated cell-to-cell signaling in root meristems to the perception of an abiotic cue. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1126 TI - Negative regulation of ABA signaling by WRKY33 is critical for Arabidopsis immunity towards Botrytis cinerea 2100 JO - eLife PY - 2015 SP - e07295 AU - Liu, S. AU - Kracher, B. AU - Ziegler, J. AU - Birkenbihl, R. P. AU - Somssich, I. E. AU - VL - 4 UR - DO - 10.7554/eLife.07295 AB - The Arabidopsis mutant wrky33 is highly susceptible to Botrytis cinerea. We identified >1680 Botrytis-induced WRKY33 binding sites associated with 1576 Arabidopsis genes. Transcriptional profiling defined 318 functional direct target genes at 14 hr post inoculation. Comparative analyses revealed that WRKY33 possesses dual functionality acting either as a repressor or as an activator in a promoter-context dependent manner. We confirmed known WRKY33 targets involved in hormone signaling and phytoalexin biosynthesis, but also uncovered a novel negative role of abscisic acid (ABA) in resistance towards B. cinerea 2100. The ABA biosynthesis genes NCED3 and NCED5 were identified as direct targets required for WRKY33-mediated resistance. Loss-of-WRKY33 function resulted in elevated ABA levels and genetic studies confirmed that WRKY33 acts upstream of NCED3/NCED5 to negatively regulate ABA biosynthesis. This study provides the first detailed view of the genome-wide contribution of a specific plant transcription factor in modulating the transcriptional network associated with plant immunity. A2 - C1 - Molecular Signal Processing ER - TY - CHAP ID - 62 TI - Specialized Plant Metabolites: Diversity and Biosynthesis T2 - Ecological Biochemistry: Environmental and Interspecies Interactions PB - PY - 2015 SP - 14-37 AU - Tissier, A. AU - Ziegler, J. AU - Vogt, T. AU - VL - UR - SN - 9783527686063 DO - 10.1002/9783527686063.ch2 AB - Plant secondary metabolites, also termed specialized plant metabolites, currently comprise more than 200 000 natural products that are all based on a few biosynthetic pathways and key primary metabolites. Some pathways like flavonoid and terpenoid biosynthesis are universally distributed in the plant kingdom, whereas others like alkaloid or cyanogenic glycoside biosynthesis are restricted to a limited set of taxa. Diversification is achieved by an array of mechanisms at the genetic and enzymatic level including gene duplications, substrate promiscuity of enzymes, cell‐specific regulatory systems, together with modularity and combinatorial aspects. Specialized metabolites reflect adaptations to a specific environment. The observed diversity illustrates the heterogeneity and multitude of ecological habitats and niches that plants have colonized so far and constitutes a reservoir of potential new metabolites that may provide adaptive advantage in the face of environmental changes. The code that connects the observed chemical diversity to this ecological diversity is largely unknown. One way to apprehend this diversity is to realize its tremendous plasticity and evolutionary potential. This chapter presents an overview of the most widespread and popular secondary metabolites, which provide a definite advantage to adapt to or to colonize a particular environment, making the boundary between the “primary” and the “secondary” old fashioned and blurry. A2 - Krauss, G.-J. & Nies, D. H., eds. C1 - Cell and Metabolic Biology; Molecular Signal Processing ER - TY - JOUR ID - 1281 TI - Simultaneous analysis of apolar phytohormones and 1-aminocyclopropan-1-carboxylic acid by high performance liquid chromatography/electrospray negative ion tandem mass spectrometry via 9-fluorenylmethoxycarbonyl chloride derivatization JO - J. Chromatogr. A PY - 2014 SP - 102-109 AU - Ziegler, J. AU - Qwegwer, J. AU - Schubert, M. AU - Erickson, J. L. AU - Schattat, M. AU - Bürstenbinder, K. AU - Grubb, C. D. AU - Abel, S. AU - VL - 1362 UR - DO - 10.1016/j.chroma.2014.08.029 AB - A strategy to detect and quantify the polar ethylene precursor 1-aminocyclopropan-1-carboxylic acid (ACC) along with the more apolar phytohormones abscisic acid (ABA), indole-3-acetic acid (IAA), jasmonic acid (JA), jasmonic acid-isoleucine conjugate (JA-Ile), 12-oxo-phytodienoic acid (OPDA), trans-zeatin, and trans-zeatin 9-riboside using a single extraction is presented. Solid phase resins commonly employed for extraction of phytohormones do not allow the recovery of ACC. We circumvent this problem by attaching an apolar group to ACC via derivatization with the amino group specific reagent 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl). Derivatization in the methanolic crude extract does not modify other phytohormones. The derivatized ACC could be purified and detected together with the more apolar phytohormones using common solid phase extraction resins and reverse phase HPLC/electrospray negative ion tandem mass spectrometry. The limit of detection was in the low nanomolar range for all phytohormones, a sensitivity sufficient to accurately determine the phytohormone levels from less than 50 mg (fresh weight) of Arabidopsis thaliana and Nicotiana benthamiana tissues. Comparison with previously published phytohormone levels and the reported changes in phytohormone levels after stress treatments confirmed the accuracy of the method. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1280 TI - Analysis of amino acids by HPLC/electrospray negative ion tandem mass spectrometry using 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) derivatization JO - Amino Acids PY - 2014 SP - 2799-2808 AU - Ziegler, J. AU - Abel, S. AU - VL - 46 UR - DO - 10.1007/s00726-014-1837-5 AB - A new method for the determination of amino acids is presented. It combines established methods for the derivatization of primary and secondary amino groups with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) with the subsequent amino acid specific detection of the derivatives by LC–ESI–MS/MS using multiple reaction monitoring (MRM). The derivatization proceeds within 5 min, and the resulting amino acid derivatives can be rapidly purified from matrix by solid-phase extraction (SPE) on HR-X resin and separated by reversed-phase HPLC. The Fmoc derivatives yield several amino acid specific fragment ions which opened the possibility to select amino acid specific MRM transitions. The method was applied to all 20 proteinogenic amino acids, and the quantification was performed using l-norvaline as standard. A limit of detection as low as 1 fmol/µl with a linear range of up to 125 pmol/µl could be obtained. Intraday and interday precisions were lower than 10 % relative standard deviations for most of the amino acids. Quantification using l-norvaline as internal standard gave very similar results compared to the quantification using deuterated amino acid as internal standards. Using this protocol, it was possible to record the amino acid profiles of only a single root from Arabidopsis thaliana seedlings and to compare it with the amino acid profiles of 20 dissected root meristems (200 μm). A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1205 TI - Agrobacterium-derived cytokinin influences plastid morphology and starch accumulation in Nicotiana benthamiana during transient assays JO - BMC Plant Biol. PY - 2014 SP - 127 AU - Erickson, J. L. AU - Ziegler, J. AU - Guevara, D. AU - Abel, S. AU - Klösgen, R. B. AU - Mathur, J. AU - Rothstein, S. J. AU - Schattat, M. H. AU - VL - 14 UR - DO - 10.1186/1471-2229-14-127 AB - BackgroundAgrobacterium tumefaciens-based transient assays have become a common tool for answering questions related to protein localization and gene expression in a cellular context. The use of these assays assumes that the transiently transformed cells are observed under relatively authentic physiological conditions and maintain ‘normal’ sub-cellular behaviour. Although this premise is widely accepted, the question of whether cellular organization and organelle morphology is altered in Agrobacterium-infiltrated cells has not been examined in detail. The first indications of an altered sub-cellular environment came from our observation that a common laboratory strain, GV3101(pMP90), caused a drastic increase in stromule frequency. Stromules, or ‘stroma-filled-tubules’ emanate from the surface of plastids and are sensitive to a variety of biotic and abiotic stresses. Starting from this observation, the goal of our experiments was to further characterize the changes to the cell resulting from short-term bacterial infestation, and to identify the factor responsible for eliciting these changes.ResultsUsing a protocol typical of transient assays we evaluated the impact of GV3101(pMP90) infiltration on chloroplast behaviour and morphology in Nicotiana benthamiana. Our experiments confirmed that GV3101(pMP90) consistently induces stromules and alters plastid position relative to the nucleus. These effects were found to be the result of strain-dependant secretion of cytokinin and its accumulation in the plant tissue. Bacterial production of the hormone was found to be dependant on the presence of a trans-zeatin synthase gene (tzs) located on the Ti plasmid of GV3101(pMP90). Bacteria-derived cytokinins were also correlated with changes to both soluble sugar level and starch accumulation.ConclusionAlthough we have chosen to focus on how transient Agrobacterium infestation alters plastid based parameters, these changes to the morphology and position of a single organelle, combined with the measured increases in sugar and starch content, suggest global changes to cell physiology. This indicates that cells visualized during transient assays may not be as ‘normal’ as was previously assumed. Our results suggest that the impact of the bacteria can be minimized by choosing Agrobacterium strains devoid of the tzs gene, but that the alterations to sub-cellular organization and cell carbohydrate status cannot be completely avoided using this strategy. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1199 TI - The DET1-COP1-HY5 Pathway Constitutes a Multipurpose Signaling Module Regulating Plant Photomorphogenesis and Thermomorphogenesis JO - Cell Rep. PY - 2014 SP - 1983-1989 AU - Delker, C. AU - Sonntag, L. AU - James, G. AU - Janitza, P. AU - Ibañez, C. AU - Ziermann, H. AU - Peterson, T. AU - Denk, K. AU - Mull, S. AU - Ziegler, J. AU - Davis, S. AU - Schneeberger, K. AU - Quint, M. AU - VL - 9 UR - DO - 10.1016/j.celrep.2014.11.043 AB - Developmental plasticity enables plants to respond to elevated ambient temperatures by adapting their shoot architecture. On the cellular level, the basic-helix-loop-helix (bHLH) transcription factor PHYTOCHROME INTERACTING FACTOR 4 (PIF4) coordinates this response by activating hormonal modules that in turn regulate growth. In addition to an unknown temperature-sensing mechanism, it is currently not understood how temperature regulates PIF4 activity. Using a forward genetic approach in Arabidopsis thaliana, we present extensive genetic evidence demonstrating that the DE-ETIOLATED 1 (DET1)-CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1)-ELONGATED HYPOCOTYL 5 (HY5)-dependent photomorphogenesis pathway transcriptionally regulates PIF4 to coordinate seedling growth in response to elevated temperature. Our findings demonstrate that two of the most prevalent environmental cues, light and temperature, share a much larger set of signaling components than previously assumed. Similar to the toolbox concept in animal embryonic patterning, multipurpose signaling modules might have evolved in plants to translate various environmental stimuli into adaptational growth processes. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1193 TI - Transposon Mutagenesis of the Plant-Associated Bacillus amyloliquefaciens ssp. plantarum FZB42 Revealed That the nfrA and RBAM17410 Genes Are Involved in Plant-Microbe-Interactions JO - PLOS ONE PY - 2014 SP - e98267 AU - Budiharjo, A. AU - Chowdhury, S. P. AU - Dietel, K. AU - Beator, B. AU - Dolgova, O. AU - Fan, B. AU - Bleiss, W. AU - Ziegler, J. AU - Schmid, M. AU - Hartmann, A. AU - Borriss, R. AU - VL - 9 UR - DO - 10.1371/journal.pone.0098267 AB - Bacillus amyloliquefaciens ssp. plantarum FZB42 represents the prototype of Gram-positive plant growth promoting and biocontrol bacteria. In this study, we applied transposon mutagenesis to generate a transposon library, which was screened for genes involved in multicellular behavior and biofilm formation on roots as a prerequisite of plant growth promoting activity. Transposon insertion sites were determined by rescue-cloning followed by DNA sequencing. As in B. subtilis, the global transcriptional regulator DegU was identified as an activator of genes necessary for swarming and biofilm formation, and the DegU-mutant of FZB42 was found impaired in efficient root colonization. Direct screening of 3,000 transposon insertion mutants for plant-growth-promotion revealed the gene products of nfrA and RBAM_017140 to be essential for beneficial effects exerted by FZB42 on plants. We analyzed the performance of GFP-labeled wild-type and transposon mutants in the colonization of lettuce roots using confocal laser scanning microscopy. While the wild-type strain heavily colonized root surfaces, the nfrA mutant did not colonize lettuce roots, although it was not impaired in growth in laboratory cultures, biofilm formation and swarming motility on agar plates. The RBAM17410 gene, occurring in only a few members of the B. subtilis species complex, was directly involved in plant growth promotion. None of the mutant strains were affected in producing the plant growth hormone auxin. We hypothesize that the nfrA gene product is essential for overcoming the stress caused by plant response towards bacterial root colonization. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1219 TI - Comparative analysis of Arabidopsis UGT74 glucosyltransferases reveals a special role of UGT74C1 in glucosinolate biosynthesis JO - Plant J. PY - 2014 SP - 92-105 AU - Grubb, C. D. AU - Zipp, B. J. AU - Kopycki, J. AU - Schubert, M. AU - Quint, M. AU - Lim, E.-K. AU - Bowles, D. J. AU - Pedras, M. S. C. AU - Abel, S. AU - VL - 79 UR - DO - 10.1111/tpj.12541 AB - The study of glucosinolates and their regulation has provided a powerful framework for the exploration of fundamental questions about the function, evolution, and ecological significance of plant natural products, but uncertainties about their metabolism remain. Previous work has identified one thiohydroximate S‐glucosyltransferase, UGT74B1, with an important role in the core pathway, but also made clear that this enzyme functions redundantly and cannot be the sole UDP‐glucose dependent glucosyltransferase (UGT) in glucosinolate synthesis. Here, we present the results of a nearly comprehensive in vitro activity screen of recombinant Arabidopsis Family 1 UGTs, which implicate other members of the UGT74 clade as candidate glucosinolate biosynthetic enzymes. Systematic genetic analysis of this clade indicates that UGT74C1 plays a special role in the synthesis of aliphatic glucosinolates, a conclusion strongly supported by phylogenetic and gene expression analyses. Finally, the ability of UGT74C1 to complement phenotypes and chemotypes of the ugt74b1‐2 knockout mutant and to express thiohydroximate UGT activity in planta provides conclusive evidence for UGT74C1 being an accessory enzyme in glucosinolate biosynthesis with a potential function during plant adaptation to environmental challenge. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1326 TI - Kinetic analysis of Arabidopsis glucosyltransferase UGT74B1 illustrates a general mechanism by which enzymes can escape product inhibition JO - Biochem. J. PY - 2013 SP - 37-46 AU - Kopycki, J. AU - Wieduwild, E. AU - Kohlschmidt, J. AU - Brandt, W. AU - Stepanova, A. AU - Alonso, J. AU - Pedras, M. S. AU - Abel, S. AU - Grubb, C. D. AU - VL - 450 UR - DO - 10.1042/BJ20121403 AB - Plant genomes encode numerous small molecule glycosyltransferases which modulate the solubility, activity, immunogenicity and/or reactivity of hormones, xenobiotics and natural products. The products of these enzymes can accumulate to very high concentrations, yet somehow avoid inhibiting their own biosynthesis. Glucosyltransferase UGT74B1 (UDP-glycosyltransferase 74B1) catalyses the penultimate step in the core biosynthetic pathway of glucosinolates, a group of natural products with important functions in plant defence against pests and pathogens. We found that mutation of the highly conserved Ser284 to leucine [wei9-1 (weak ethylene insensitive)] caused only very mild morphological and metabolic phenotypes, in dramatic contrast with knockout mutants, indicating that steady state glucosinolate levels are actively regulated even in unchallenged plants. Analysis of the effects of the mutation via a structural modelling approach indicated that the affected serine interacts directly with UDP-glucose, but also predicted alterations in acceptor substrate affinity and the kcat value, sparking an interest in the kinetic behaviour of the wild-type enzyme. Initial velocity and inhibition studies revealed that UGT74B1 is not inhibited by its glycoside product. Together with the effects of the missense mutation, these findings are most consistent with a partial rapid equilibrium ordered mechanism. This model explains the lack of product inhibition observed both in vitro and in vivo, illustrating a general mechanism whereby enzymes can continue to function even at very high product/precursor ratios. A2 - C1 - Bioorganic Chemistry; Molecular Signal Processing ER - TY - JOUR ID - 1295 TI - Arabidopsis Calmodulin-binding Protein IQ67-Domain 1 Localizes to Microtubules and Interacts with Kinesin Light Chain-related Protein-1 JO - J. Biol. Chem. PY - 2013 SP - 1871-1882 AU - Bürstenbinder, K. AU - Savchenko, T. AU - Müller, J. AU - Adamson, A. W. AU - Stamm, G. AU - Kwong, R. AU - Zipp, B. J. AU - Dinesh, D. C. AU - Abel, S. AU - VL - 288 UR - DO - 10.1074/jbc.M112.396200 AB - Calcium (Ca2+) is a key second messenger in eukaryotes and regulates diverse cellular processes, most notably via calmodulin (CaM). In Arabidopsis thaliana, IQD1 (IQ67 domain 1) is the founding member of the IQD family of putative CaM targets. The 33 predicted IQD proteins share a conserved domain of 67 amino acids that is characterized by a unique arrangement of multiple CaM recruitment motifs, including so-called IQ motifs. Whereas IQD1 has been implicated in the regulation of defense metabolism, the biochemical functions of IQD proteins remain to be elucidated. In this study we show that IQD1 binds to multiple Arabidopsis CaM and CaM-like (CML) proteins in vitro and in yeast two-hybrid interaction assays. CaM overlay assays revealed moderate affinity of IQD1 to CaM2 (Kd ∼ 0.6 μm). Deletion mapping of IQD1 demonstrated the importance of the IQ67 domain for CaM2 binding in vitro, which is corroborated by interaction of the shortest IQD member, IQD20, with Arabidopsis CaM/CMLs in yeast. A genetic screen of a cDNA library identified Arabidopsis kinesin light chain-related protein-1 (KLCR1) as an IQD1 interactor. The subcellular localization of GFP-tagged IQD1 proteins to microtubules and the cell nucleus in transiently and stably transformed plant tissues (tobacco leaves and Arabidopsis seedlings) suggests direct interaction of IQD1 and KLCR1 in planta that is supported by GFP∼IQD1-dependent recruitment of RFP∼KLCR1 and RFP∼CaM2 to microtubules. Collectively, the prospect arises that IQD1 and related proteins provide Ca2+/CaM-regulated scaffolds for facilitating cellular transport of specific cargo along microtubular tracks via kinesin motor proteins. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1284 TI - The emerging function of IQD proteins as scaffolds in cellular signaling and trafficking JO - Plant Signal Behav. PY - 2013 SP - e24369 AU - Abel, S. AU - Bürstenbinder, K. AU - Müller, J. AU - VL - 8 UR - DO - 10.4161/psb.24369 AB - Calcium (Ca2+) signaling modules are essential for adjusting plant growth and performance to environmental constraints. Differential interactions between sensors of Ca2+ dynamics and their molecular targets are at the center of the transduction process. Calmodulin (CaM) and CaM-like (CML) proteins are principal Ca2+-sensors in plants that govern the activities of numerous downstream proteins with regulatory properties. The families of IQ67-Domain (IQD) proteins are a large class of plant-specific CaM/CML-targets (e.g., 33 members in A. thaliana) which share a unique domain of multiple varied CaM retention motifs in tandem orientation. Genetic studies in Arabidopsis and tomato revealed first roles for IQD proteins related to basal defense response and plant development. Molecular, biochemical and histochemical analysis of Arabidopsis IQD1 demonstrated association with microtubules as well as targeting to the cell nucleus and nucleolus. In vivo binding to CaM and kinesin light chain-related protein-1 (KLCR1) suggests a Ca2+-regulated scaffolding function of IQD1 in kinesin motor-dependent transport of multiprotein complexes. Furthermore, because IQD1 interacts in vitro with single-stranded nucleic acids, the prospect arises that IQD1 and other IQD family members facilitate cellular RNA localization as one mechanism to control and fine-tune gene expression and protein sorting. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1400 TI - Polyamine homeostasis in wild type and phenolamide deficient Arabidopsis thaliana stamens JO - Front. Plant Sci. PY - 2012 SP - 180 AU - Fellenberg, C. AU - Ziegler, J. AU - Handrick, V. AU - Vogt, T. AU - VL - 3 UR - DO - 10.3389/fpls.2012.00180 AB - Polyamines (PAs) like putrescine, spermidine, and spermine are ubiquitous polycationic molecules that occur in all living cells and have a role in a wide variety of biological processes. High amounts of spermidine conjugated to hydroxycinnamic acids are detected in the tryphine of Arabidopsis thaliana pollen grains. Tapetum localized spermidine hydroxycinnamic acid transferase (SHT) is essential for the biosynthesis of these anther specific tris-conjugated spermidine derivatives. Sht knockout lines show a strong reduction of hydroxycinnamic acid amides (HCAAs). The effect of HCAA-deficient anthers on the level of free PAs was measured by a new sensitive and reproducible method using 9-fluorenylmethyl chloroformate (FMOC) and fluorescence detection by HPLC. PA concentrations can be accurately determined even when very limited amounts of plant material, as in the case of A. thaliana stamens, are available. Analysis of free PAs in wild type stamens compared to sht deficient mutants and transcript levels of key PA biosynthetic genes revealed a highly controlled regulation of PA homeostasis in A. thaliana anthers. A2 - C1 - Molecular Signal Processing; Cell and Metabolic Biology ER - TY - JOUR ID - 1499 TI - Chemoenzymatic synthesis of diverse thiohydroximates from glucosinolate-utilizing enzymes from Helix pomatia and Caldicellulosiruptor saccharolyticus JO - Biotechnol. Lett. PY - 2011 SP - 1039-1046 AU - Kopycki, J. AU - Schmidt, J. AU - Abel, S. AU - Grubb, C. D. AU - VL - 33 UR - DO - 10.1007/s10529-011-0530-y AB - Thiohydroximates comprise a diverse class of compounds important in both biological and industrial chemistry. Their syntheses are generally limited to simple alkyl and aryl compounds with few stereocenters and a narrow range of functional groups. We hypothesized that sequential action of two recombinant enzymes, a sulfatase from Helix pomatia and a β-O-glucosidase from Caldicellulosiruptor saccharolyticus, on glucosinolates would allow synthesis of thiohydroximates from a structurally broad array of abundant precursors. We report successful synthesis of thiohydroximates of varied chemical classes, including from homochiral compounds of demonstrated biological activity. The chemoenzymatic synthetic route reported here should allow access to many, if not all, of the thiohydroximate core structures of the ~200 known naturally occurring glucosinolates. The enrichment of this group for compounds with possible pharmacological potential is discussed. A2 - C1 - Molecular Signal Processing; Bioorganic Chemistry ER - TY - JOUR ID - 1478 TI - Phosphorus and nitrogen interaction: loss of QC identity in response to P or N limitation is antecipated in pdr23 mutant JO - Braz. J. Plant Physiol. PY - 2011 SP - 219-229 AU - Costa, C. T. AU - Strieder, M. L. AU - Abel, S. AU - Delatorre, C. A. AU - VL - 23 UR - DO - 10.1590/S1677-04202011000300006 AB - Changes in root architecture are an important adaptive strategy used by plants in response to limited nutrient availability to increase the odds of acquiring them. The quiescent center (QC) plays an important role by altering the meristem activity causing differentiation and therefore, inducing a determinate growth program. The arabidopsis mutant pdr23 presents primary short root in the presence of nitrate and is inefficient in the use of nucleic acids as a source of phosphorus. In this study the effect of the pdr23 mutation on the QC maintenance under low phosphorus (P) and/or nitrogen is evaluated. QC identity is maintained in wild-type in the absence of nitrate and/or phosphate if nucleic acids can be used as an alternative source of these nutrients, but not in pdr23. The mutant is not able to use nucleic acids efficiently for substitute Pi, determinate growth is observed, similar to wild-type in the total absence of P. In the absence of N pdr23 loses the expression of QC identity marker earlier than wild-type, indicating that not only the response to P is altered, but also to N. The data suggest that the mutation affects a gene involved either in the crosstalk between these nutrients or in a pathway shared by both nutrients limitation response. Moreover loss of QC identity is also observed in wild-type in the absence of N at longer limitation. Less drastic symptoms are observed in lateral roots of both genotypes. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1467 TI - Phosphate sensing in root development JO - Curr. Opin. Plant Biol. PY - 2011 SP - 303-309 AU - Abel, S. AU - VL - 14 UR - DO - 10.1016/j.pbi.2011.04.007 AB - Phosphate (Pi) and its anhydrides constitute major nodes in metabolism. Thus, plant performance depends directly on Pi nutrition. Inadequate Pi availability in the rhizosphere is a common challenge to plants, which activate metabolic and developmental responses to maximize Pi usage and acquisition. The sensory mechanisms that monitor environmental Pi and transmit the nutritional signal to adjust root development have increasingly come into focus. Recent transcriptomic analyses and genetic approaches have highlighted complex antagonistic interactions between external Pi and Fe bioavailability and have implicated the stem cell niche as a target of Pi sensing to regulate root meristem activity. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1530 TI - Odyssey of Auxin JO - Cold Spring Harb. Perspect. Biol. PY - 2010 SP - a004572 AU - Abel, S. AU - Theologis, A. AU - VL - 2 UR - DO - 10.1101/cshperspect.a004572 AB - The history of plant biology is inexorably intertwined with the conception and discovery of auxin, followed by the many decades of research to comprehend its action during growth and development. Growth responses to auxin are complex and require the coordination of auxin production, transport, and perception. In this overview of past auxin research, we limit our discourse to the mechanism of auxin action. We attempt to trace the almost epic voyage from the birth of the hormonal concept in plants to the recent crystallographic studies that resolved the TIR1-auxin receptor complex, the first structural model of a plant hormone receptor. The century-long endeavor is a beautiful illustration of the power of scientific reasoning and human intuition, but it also brings to light the fact that decisive progress is made when new technologies emerge and disciplines unite. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1662 TI - ER-resident proteins PDR2 and LPR1 mediate the developmental response of root meristems to phosphate availability JO - Proc. Natl. Acad. Sci. U.S.A. PY - 2009 SP - 14174-14179 AU - Ticconi, C. A. AU - Lucero, R. D. AU - Sakhonwasee, S. AU - Adamson, A. W. AU - Creff, A. AU - Nussaume, L. AU - Desnos, T. AU - Abel, S. AU - VL - 106 UR - DO - 10.1073/pnas.0901778106 AB - Inadequate availability of inorganic phosphate (Pi) in the rhizosphere is a common challenge to plants, which activate metabolic and developmental responses to maximize Pi acquisition. The sensory mechanisms that monitor environmental Pi status and regulate root growth via altered meristem activity are unknown. Here, we show that phosphate deficiency response 2 (PDR2) encodes the single P5-type ATPase of Arabidopsis thaliana. PDR2 functions in the endoplasmic reticulum (ER) and is required for proper expression of scarecrow (SCR), a key regulator of root patterning, and for stem-cell maintenance in Pi-deprived roots. We further show that the multicopper oxidase encoded by low phosphate root 1 (LPR1) is targeted to the ER and that LPR1 and PDR2 interact genetically. Because the expression domains of both genes overlap in the stem-cell niche and distal root meristem, we propose that PDR2 and LPR1 function together in an ER-resident pathway that adjusts root meristem activity to external Pi. Our data indicate that the Pi-conditional root phenotype of pdr2 is not caused by increased Fe availability in low Pi; however, Fe homeostasis modifies the developmental response of root meristems to Pi availability. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1761 TI - Auxin Is Surfacing JO - ACS Chem. Biol. PY - 2007 SP - 380-384 AU - Abel, S. AU - VL - 2 UR - DO - 10.1021/cb7001158 AB - Indole-3-acetic acid (IAA or auxin) is essential throughout the life cycle of a plant. It controls diverse cellular processes, including gene expression. The hormone is perceived by a ubiquitin protein ligase (E3) and triggers the rapid destruction of repressors, called Aux/IAA proteins. The first structural model of a plant hormone receptor illustrates how auxin promotes Aux/IAA substrate recruitment by extending the hydrophobic protein-interaction surface. This work establishes a novel mechanism of E3 regulation by small molecules and promises a novel strategy for the treatment of human disorders associated with defective ubiquitin-dependent proteolysis. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1869 TI - Glucosinolate metabolism and its control JO - Trends Plant Sci. PY - 2006 SP - 89-100 AU - Grubb, C. D. AU - Abel, S. AU - VL - 11 UR - DO - 10.1016/j.tplants.2005.12.006 AB - Glucosinolates and their associated degradation products have long been recognized for their distinctive benefits to human nutrition and plant defense. Because most of the structural genes of glucosinolate metabolism have been identified and functionally characterized in Arabidopsis thaliana, current research increasingly focuses on questions related to the regulation of glucosinolate synthesis, distribution and degradation as well as to the feasibility of engineering customized glucosinolate profiles. Here, we highlight recent progress in glucosinolate research, with particular emphasis on the biosynthetic pathway and its metabolic relationships to auxin homeostasis. We further discuss emerging insight into the signaling networks and regulatory proteins that control glucosinolate accumulation during plant development and in response to environmental challenge. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1965 TI - Transient Agrobacterium-mediated gene expression in the Arabidopsis hydroponics root system for subcellular localization studies JO - Plant Mol. Biol. Rep. PY - 2005 SP - 179-184 AU - Levy, M. AU - Rachmilevitch, S. AU - Abel, S. AU - VL - 23 UR - DO - 10.1007/BF02772708 AB - To a great extent, the cellular compartmentalization and molecular interactions are indicative of the function of a protein. The development of simple and efficient tools for testing the subcellular location of proteins is indispensable to elucidate the function of genes in plants. In this report, we assessed the feasibility ofAgrobacterium-mediated transformation of hydroponically grown roots to follow intracellular targeting of proteins fused to green fluorescent protein (GFP). We developed a simple in planta assay for subcellular localization of proteins inArabidopsis roots via transient transformation and tested this method by expressing a GFP fusion of a known nuclear protein, IQD1. Visualization of transiently expressed GFP fusion proteins in roots by means of confocal microscopy is superior to the analysis of green tissues because the roots are virtually transparent and free of chlorophyll autofluorescence. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1964 TI - Arabidopsis IQD1, a novel calmodulin-binding nuclear protein, stimulates glucosinolate accumulation and plant defense JO - Plant J. PY - 2005 SP - 79-96 AU - Levy, M. AU - Wang, Q. AU - Kaspi, R. AU - Parrella, M. P. AU - Abel, S. AU - VL - 43 UR - DO - 10.1111/j.1365-313X.2005.02435.x AB - Glucosinolates are a class of secondary metabolites with important roles in plant defense and human nutrition. To uncover regulatory mechanisms of glucosinolate production, we screened Arabidopsis thaliana T‐DNA activation‐tagged lines and identified a high‐glucosinolate mutant caused by overexpression of IQD1 (At3g09710). A series of gain‐ and loss‐of‐function IQD1 alleles in different accessions correlates with increased and decreased glucosinolate levels, respectively. IQD1 encodes a novel protein that contains putative nuclear localization signals and several motifs known to mediate calmodulin binding, which are arranged in a plant‐specific segment of 67 amino acids, called the IQ67 domain. We demonstrate that an IQD1‐GFP fusion protein is targeted to the cell nucleus and that recombinant IQD1 binds to calmodulin in a Ca2+‐dependent fashion. Analysis of steady‐state messenger RNA levels of glucosinolate pathway genes indicates that IQD1 affects expression of multiple genes with roles in glucosinolate metabolism. Histochemical analysis of tissue‐specific IQD1 ::GUS expression reveals IQD1 promoter activity mainly in vascular tissues of all organs, consistent with the expression patterns of several glucosinolate‐related genes. Interestingly, overexpression of IQD1 reduces insect herbivory, which we demonstrated in dual‐choice assays with the generalist phloem‐feeding green peach aphid (Myzus persicae ), and in weight‐gain assays with the cabbage looper (Trichoplusia ni ), a generalist‐chewing lepidopteran. As IQD1 is induced by mechanical stimuli, we propose IQD1 to be novel nuclear factor that integrates intracellular Ca2+ signals to fine‐tune glucosinolate accumulation in response to biotic challenge. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1944 TI - Animal Models and Analytical Approaches for Understanding the Relationships Between Wine and Cancer JO - Drugs Exp. Clin. Res. PY - 2005 SP - 19-27 AU - Ebeler, S. E. AU - Dingley, K. H. AU - Ubick, E. AU - Abel, S. AU - Mitchell, A. E. AU - Burns, S. A. AU - Steinberg, F. M. AU - Clifford, A. J. AU - VL - 31 UR - https://pubmed.ncbi.nlm.nih.gov/15921026/ AB - We used two approaches for studying the relationships between wine consumption, wine composition and cancer In the first approach, a transgenic mouse model of human neurofibromatosis, combined with the use of well-defined, chemically purified diets, showed that red wine contains nonalcoholic components that can delay tumor onset. In additional studies, catechin, the main monomeric polyphenol of red wine, delayed tumor onset in this mouse model in a positive, linear relationship when incorporated into the diet at levels of 0.5-4 mmol/kg diet. In the second approach, low doses of the chemical carcinogen 2-amino-1-methyl-6-phenylimidazo(4, 5-b)pyridine (PhlP) were administered to rats, and formation of DNA adducts was evaluated by accelerator mass spectrometry. Consumption of red wine solids (the residue from red wine remaining after removal of alcohol and water) and the wine polyphenol quercetin did not influence PhlP-DNA adduct levels or induce liver enzymes (glutathione-S-transferase and quinone reductase). However, quercetin did alter distribution of PhlP in the rat tissues compared to control animals and animals fed other potential dietary chemopreventive agents, including phenylethyl isothiocyanate and sulforaphane. These studies demonstrate the feasibility of these approaches for studying the chemopreventive potential of dietary components at physiologic levels in A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 1929 TI - Genome-wide comparative analysis of the IQD gene families in Arabidopsis thaliana and Oryza sativa JO - BMC Evol. Biol. PY - 2005 SP - 72 AU - Abel, S. AU - Savchenko, T. AU - Levy, M. AU - VL - 5 UR - DO - 10.1186/1471-2148-5-72 AB - BackgroundCalcium signaling plays a prominent role in plants for coordinating a wide range of developmental processes and responses to environmental cues. Stimulus-specific generation of intracellular calcium transients, decoding of calcium signatures, and transformation of the signal into cellular responses are integral modules of the transduction process. Several hundred proteins with functions in calcium signaling circuits have been identified, and the number of downstream targets of calcium sensors is expected to increase. We previously identified a novel, calmodulin-binding nuclear protein, IQD1, which stimulates glucosinolate accumulation and plant defense in Arabidopsis thaliana. Here, we present a comparative genome-wide analysis of a new class of putative calmodulin target proteins in Arabidopsis and rice.ResultsWe identified and analyzed 33 and 29 IQD1-like genes in Arabidopsis thaliana and Oryza sativa, respectively. The encoded IQD proteins contain a plant-specific domain of 67 conserved amino acid residues, referred to as the IQ67 domain, which is characterized by a unique and repetitive arrangement of three different calmodulin recruitment motifs, known as the IQ, 1-5-10, and 1-8-14 motifs. We demonstrated calmodulin binding for IQD20, the smallest IQD protein in Arabidopsis, which consists of a C-terminal IQ67 domain and a short N-terminal extension. A striking feature of IQD proteins is the high isoelectric point (~10.3) and frequency of serine residues (~11%). We compared the Arabidopsis and rice IQD gene families in terms of gene structure, chromosome location, predicted protein properties and motifs, phylogenetic relationships, and evolutionary history. The existence of an IQD-like gene in bryophytes suggests that IQD proteins are an ancient family of calmodulin-binding proteins and arose during the early evolution of land plants.ConclusionComparative phylogenetic analyses indicate that the major IQD gene lineages originated before the monocot-eudicot divergence. The extant IQD loci in Arabidopsis primarily resulted from segmental duplication and reflect preferential retention of paralogous genes, which is characteristic for proteins with regulatory functions. Interaction of IQD1 and IQD20 with calmodulin and the presence of predicted calmodulin binding sites in all IQD family members suggest that IQD proteins are a new class of calmodulin targets. The basic isoelectric point of IQD proteins and their frequently predicted nuclear localization suggest that IQD proteins link calcium signaling pathways to the regulation of gene expression. Our comparative genomics analysis of IQD genes and encoded proteins in two model plant species provides the first step towards the functional dissection of this emerging family of putative calmodulin targets. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2057 TI - Short on phosphate: plant surveillance and countermeasures JO - Trends Plant Sci. PY - 2004 SP - 548-555 AU - Ticconi, C. A. AU - Abel, S. AU - VL - 9 UR - DO - 10.1016/j.tplants.2004.09.003 AB - Metabolism depends on inorganic phosphate (Pi) as reactant, allosteric effector and regulatory moiety in covalent protein modification. To cope with Pi shortage (a common situation in many ecosystems), plants activate a set of adaptive responses to enhance Pi recycling and acquisition by reprogramming metabolism and restructuring root system architecture. The physiology of Pi starvation responses has become well understood, and so current research focuses on the initial molecular events that sense, transmit and integrate information about external and internal Pi status. Recent studies have provided evidence for Pi as a signaling molecule and initial insight into the coordination of Pi deficiency responses at the cellular and molecular level. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2056 TI - Arabidopsis pdr2 reveals a phosphate-sensitive checkpoint in root development JO - Plant J. PY - 2004 SP - 801-814 AU - Ticconi, C. A. AU - Delatorre, C. A. AU - Lahner, B. AU - Salt, D. E. AU - Abel, S. AU - VL - 37 UR - DO - 10.1111/j.1365-313x.2004.02005.x AB - Plants have evolved complex strategies to maintain phosphate (Pi) homeostasis and to maximize Pi acquisition when the macronutrient is limiting. Adjustment of root system architecture via changes in meristem initiation and activity is integral to the acclimation process. However, the mechanisms that monitor external Pi status and interpret the nutritional signal remain to be elucidated. Here, we present evidence that the Pi deficiency response , pdr2 , mutation disrupts local Pi sensing. The sensitivity and amplitude of metabolic Pi‐starvation responses, such as Pi‐responsive gene expression or accumulation of anthocyanins and starch, are enhanced in pdr2 seedlings. However, the most conspicuous alteration of pdr2 is a conditional short‐root phenotype that is specific for Pi deficiency and caused by selective inhibition of root cell division followed by cell death below a threshold concentration of about 0.1 mm external Pi. Measurements of general Pi uptake and of total phosphorus (P) in root tips exclude a defect in high‐affinity Pi acquisition. Rescue of root meristem activity in Pi‐starved pdr2 by phosphite (Phi), a non‐metabolizable Pi analog, and divided‐root experiments suggest that pdr2 disrupts sensing of low external Pi availability. Thus, PDR2 is proposed to function at a Pi‐sensitive checkpoint in root development, which monitors environmental Pi status, maintains and fine‐tunes meristematic activity, and finally adjusts root system architecture to maximize Pi acquisition. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2019 TI - Arabidopsis glucosyltransferase UGT74B1 functions in glucosinolate biosynthesis and auxin homeostasis JO - Plant J. PY - 2004 SP - 893-908 AU - Grubb, C. D. AU - Zipp, B. J. AU - Ludwig-Müller, J. AU - Masuno, M. N. AU - Molinski, T. F. AU - Abel, S. AU - VL - 40 UR - DO - 10.1111/j.1365-313X.2004.02261.x AB - Glucosinolates are a class of secondary metabolites with important roles in plant defense and human nutrition. Here, we characterize a putative UDP‐glucose:thiohydroximate S‐glucosyltransferase, UGT74B1, to determine its role in the Arabidopsis glucosinolate pathway. Biochemical analyses demonstrate that recombinant UGT74B1 specifically glucosylates the thiohydroximate functional group. Low K m values for phenylacetothiohydroximic acid (approximately 6 μ m ) and UDP‐glucose (approximately 50 μm ) strongly suggest that thiohydroximates are in vivo substrates of UGT74B1. Insertional loss‐of‐function ugt74b1 mutants exhibit significantly decreased, but not abolished, glucosinolate accumulation. In addition, ugt74b1 mutants display phenotypes reminiscent of auxin overproduction, such as epinastic cotyledons, elongated hypocotyls in light‐grown plants, excess adventitious rooting and incomplete leaf vascularization. Indeed, during early plant development, mutant ugt74b1 seedlings accumulate nearly threefold more indole‐3‐acetic acid than the wild type. Other phenotypes, however, such as chlorosis along the leaf veins, are likely caused by thiohydroximate toxicity. Analysis of UGT74B1 promoter activity during plant development reveals expression patterns consistent with glucosinolate metabolism and induction by auxin treatment. The results are discussed in the context of known mutations in glucosinolate pathway genes and their effects on auxin homeostasis. Taken together, our work provides complementary in vitro and in vivo evidence for a primary role of UGT74B1 in glucosinolate biosynthesis. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2080 TI - Effect of Dietary Constituents With Chemopreventive Potential on Adduct Formation of a Low Dose of the Heterocyclic Amines PhIP and IQ and Phase II Hepatic Enzymes JO - Nutr. Cancer PY - 2003 SP - 212-221 AU - Dingley, K. H. AU - Ubick, E. A. AU - Chiarappa-Zucca, M. L. AU - Nowell, S. AU - Abel, S. AU - Ebeler, S. E. AU - Mitchell, A. E. AU - Burns, S. A. AU - Steinberg, F. M. AU - Clifford, A. J. AU - VL - 46 UR - DO - 10.1207/S15327914NC4602_15 AB - We conducted a study to evaluate dietary chemopreventive strategies to reduce genotoxic effects of the carcinogens 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) and 2-amino-3-methylimidazo[4,5-f]quinoline (IQ). PhIP and IQ are heterocyclic amines (HCAs) that are found in cooked meat and may be risk factors for cancer. Typical chemoprevention studies have used carcinogen doses many thousand-fold higher than usual human daily intake. Therefore, we administered a low dose of [14C] PhIPand [3H] IQand utilized accelerator mass spectrometry to quantify PhIP adducts in the liver, colon, prostate, and blood plasma and IQadducts in the liver and blood plasma with high sensitivity. Diets supplemented with phenethylisothiocyanate (PEITC), genistein, chlorophyllin, or lycopene were evaluated for their ability to decrease adduct formation of [14C] PhIPand [3H] IQin rats. We also examined the effect of treatments on the activity of the phase II detoxification enzymes glutathione S-transferase (GST), UDP-glucuronyltransferase (UGT), phenol sulfotransferase (SULT) and quinone reductase (QR). PEITC and chlorophyllin significantly decreased PhIP-DNA adduct levels in all tissues examined, which was reflected by similar changes in PhIP binding to albumin in the blood. In contrast, genistein and lycopene tended to increase PhIP adduct levels. The treatments did not significantly alter the level of IQ-DNA or -protein adducts in the liver.With the exception of lycopene, the treatments had some effect on the activity of one or more hepatic phase II detoxification enzymes. We conclude that PEITC and chlorophyllin are protective of PhIP-induced genotoxicity after a low exposure dose of carcinogen, possibly through modification of HCA metabolism. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2174 TI - FQR1, a Novel Primary Auxin-Response Gene, Encodes a Flavin Mononucleotide-Binding Quinone Reductase JO - Plant Physiol. PY - 2002 SP - 578-590 AU - Laskowski, M. J. AU - Dreher, K. A. AU - Gehring, M. A. AU - Abel, S. AU - Gensler, A. L. AU - Sussex, I. M. AU - VL - 128 UR - DO - 10.1104/pp.010581 AB - FQR1 is a novel primary auxin-response gene that codes for a flavin mononucleotide-binding flavodoxin-like quinone reductase. Accumulation of FQR1 mRNA begins within 10 min of indole-3-acetic acid application and reaches a maximum of approximately 10-fold induction 30 min after treatment. This increase in FQR1 mRNA abundance is not diminished by the protein synthesis inhibitor cycloheximide, demonstrating thatFQR1 is a primary auxin-response gene. Sequence analysis reveals that FQR1 belongs to a family of flavin mononucleotide-binding quinone reductases. Partially purified His-tagged FQR1 isolated fromEscherichia coli catalyzes the transfer of electrons from NADH and NADPH to several substrates and exhibits in vitro quinone reductase activity. Overexpression of FQR1 in plants leads to increased levels of FQR1 protein and quinone reductase activity, indicating that FQR1 functions as a quinone reductase in vivo. In mammalian systems, glutathione S-transferases and quinone reductases are classified as phase II detoxification enzymes. We hypothesize that the auxin-inducible glutathioneS-transferases and quinone reductases found in plants also act as detoxification enzymes, possibly to protect against auxin-induced oxidative stress. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2162 TI - Identification of Arabidopsis mutants with altered glucosinolate profiles based on isothiocyanate bioactivity JO - Plant Sci. PY - 2002 SP - 143-152 AU - Grubb, C. D. AU - Gross, H. B. AU - Chen, D. L. AU - Abel, S. AU - VL - 162 UR - DO - 10.1016/S0168-9452(01)00550-7 AB - Glucosinolates are a diverse class of nitrogen- and sulfur-containing secondary metabolites. They are rapidly hydrolyzed on tissue disruption to a number of biologically active compounds that are increasingly attracting interest as anticarcinogenic phytochemicals and crop protectants. Several glucosinolate-derived isothiocyanates are potent chemopreventive agents that favorably modulate carcinogen metabolism in mammals. Methylsulfinylalkyl isothiocyanates, in particular the 4-methylsulfinylbutyl derivative, are selective and potent inducers of mammalian detoxification enzymes such as quinone reductase (QR). Cruciferous plants including Arabidopsis thaliana (L.) Heyhn, synthesize methylsulfinylalkyl glucosinolates, which are derived from methionine. Using a colorimetric assay for QR activity in murine hepatoma cells and high performance liquid chromatography (HPLC) analysis of desulfoglucosinolates, we have demonstrated a strong positive correlation between leaf QR inducer potency and leaf content of methionine-derived glucosinolates in various A. thaliana ecotypes and available glucosinolate mutants. In a molecular genetic approach to glucosinolate biosynthesis, we screened 3000 chemically mutagenized M2 plants of the Columbia ecotype for altered leaf QR inducer potency. Subsequent HPLC analysis of progeny of putative mutants identified six lines with significant and heritable changes in leaf glucosinolate content and composition. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2195 TI - Direct analysis of single leaf disks for chemopreventive glucosinolates JO - Phytochem. Anal. PY - 2002 SP - 152-157 AU - Wang, Q. AU - Grubb, C. D. AU - Abel, S. AU - VL - 13 UR - DO - 10.1002/pca.636 AB - Natural isothiocyanates, produced during plant tissue damage from methionine‐derived glucosinolates, are potent inducers of mammalian phase 2 detoxification enzymes such as quinone reductase (QR). A greatly simplified bioassay for glucosinolates based on induction and colorimetric detection of QR activity in murine hepatoma cells is described. It is demonstrated that excised leaf disks of Arabidopsis thaliana (ecotype Columbia) can directly and reproducibly substitute for cell‐free leaf extracts as inducers of murine QR, which reduces sample preparation to a minimum and maximizes throughput. A comparison of 1 and 3 mm diameter leaf disks indicated that QR inducer potency was proportional to disk circumference (extent of tissue damage) rather than to area. When compared to the QR inducer potency of the corresponding amount of extract, 1 mm leaf disks were equally effective, whereas 3 mm disks were 70% as potent. The QR inducer potency of leaf disks correlated positively with the content of methionine‐derived glucosinolates, as shown by the analysis of wild‐type plants and mutant lines with lower or higher glucosinolate content. Thus, the microtitre plate‐based assay of single leaf disks provides a robust and inexpensive visual method for rapidly screening large numbers of plants in mapping populations or mutant collections and may be applicable to other glucosinolate‐producing species. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2134 TI - Phosphate sensing in higher plants JO - Physiol. Plant. PY - 2002 SP - 1-8 AU - Abel, S. AU - Ticconi, C. A. AU - Delatorre, C. A. AU - VL - 115 UR - DO - 10.1034/j.1399-3054.2002.1150101.x AB - Phosphate (Pi) plays a central role as reactant and effector molecule in plant cell metabolism. However, Pi is the least accessible macronutrient in many ecosystems and its low availability often limits plant growth. Plants have evolved an array of molecular and morphological adaptations to cope with Pi limitation, which include dramatic changes in gene expression and root development to facilitate Pi acquisition and recycling. Although physiological responses to Pi starvation have been increasingly studied and understood, the initial molecular events that monitor and transmit information on external and internal Pi status remain to be elucidated in plants. This review summarizes molecular and developmental Pi starvation responses of higher plants and the evidence for coordinated regulation of gene expression, followed by a discussion of the potential involvement of plant hormones in Pi sensing and of molecular genetic approaches to elucidate plant signalling of low Pi availability. Complementary genetic strategies in Arabidopsis thaliana have been developed that are expected to identify components of plant signal transduction pathways involved in Pi sensing. Innovative screening methods utilize reporter gene constructs, conditional growth on organophosphates and the inhibitory properties of the Pi analogue phosphite, which hold the promise for significant advances in our understanding of the complex mechanisms by which plants regulate Pi‐starvation responses. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2239 TI - Attenuation of Phosphate Starvation Responses by Phosphite in Arabidopsis JO - Plant Physiol. PY - 2001 SP - 963-972 AU - Ticconi, C. A. AU - Delatorre, C. A. AU - Abel, S. AU - VL - 127 UR - DO - 10.1104/pp.010396 AB - When inorganic phosphate is limiting, Arabidopsis has the facultative ability to metabolize exogenous nucleic acid substrates, which we utilized previously to identify insensitive phosphate starvation response mutants in a conditional genetic screen. In this study, we examined the effect of the phosphate analog, phosphite (Phi), on molecular and morphological responses to phosphate starvation. Phi significantly inhibited plant growth on phosphate-sufficient (2 mm) and nucleic acid-containing (2 mmphosphorus) media at concentrations higher than 2.5 mm. However, with respect to suppressing typical responses to phosphate limitation, Phi effects were very similar to those of phosphate. Phosphate starvation responses, which we examined and found to be almost identically affected by both anions, included changes in: (a) the root-to-shoot ratio; (b) root hair formation; (c) anthocyanin accumulation; (d) the activities of phosphate starvation-inducible nucleolytic enzymes, including ribonuclease, phosphodiesterase, and acid phosphatase; and (e) steady-state mRNA levels of phosphate starvation-inducible genes. It is important that induction of primary auxin response genes by indole-3-acetic acid in the presence of growth-inhibitory Phi concentrations suggests that Phi selectively inhibits phosphate starvation responses. Thus, the use of Phi may allow further dissection of phosphate signaling by genetic selection for constitutive phosphate starvation response mutants on media containing organophosphates as the only source of phosphorus. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2227 TI - Inheritance of Three Major Genes Involved in the Synthesis of Aliphatic Glucosinolates in Brassica oleracea JO - J. Am. Soc. Hortic. Sci. PY - 2001 SP - 427-431 AU - Li, G. AU - Riaz, A. AU - Goyal, S. AU - Abel, S. AU - Quiros, C. F. AU - VL - 126 UR - DO - 10.21273/JASHS.126.4.427 AB - Inheritance of three major genes involved in synthesis of aliphatic glucosinolates (GSL) was followed in segregating populations of Brassica oleracea L. generated from three crosses: broccoli × cauliflower, collard × broccoli, and collard × cauliflower. Two of these genes, GSL-PRO and GSL-ELONG, regulate sidechain length. The action of the former results in three-carbon GSL, whereas action of the latter produces four-carbon GSL. We determined that these two genes act and segregate independently from each other in B. oleracea. The double recessive genotype produces only trace amounts of aliphatic GSL. The third gene, GSL-ALK controls sidechain desaturation and, as it has been observed in Arabidopsis thaliana (L.) Heynh., we found that this gene cosegregates with a fourth gene, GSL-OH, that is responsible for sidechain hydroxylation. Elucidation of the inheritance of major genes controlling biosynthesis of GSL will allow for manipulation of these genes and facilitate development of lines with specific GSL profiles. This capability will be important for improvement of Brassica breeding lines with high content of desirable GSL, like glucoraphanin, a demonstrated precursor of anticarcinogenic compounds. Additionally, this work is the first step towards cloning the major genes of the aliphatic GSL pathway, and to use these clones in transformation strategies for further crop enhancement. A2 - C1 - Molecular Signal Processing ER - TY - CHAP ID - 161 TI - Secretory Acid Ribonucleases from Tomato, Lycopersicon esculentum Mill. T2 - Ribonucleases - Part A PB - Methods Enzymol. PY - 2001 SP - 351-368 AU - Abel, S. AU - Köck, M. AU - VL - 341 UR - DO - 10.1016/S0076-6879(01)41163-3 AB - A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2255 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 - 2253 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 - 2252 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 - 2244 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 - 2320 TI - Biochemical characterization of recombinant polypeptides corresponding to the predicted βαα fold in Aux/IAA proteins JO - FEBS Lett. PY - 1999 SP - 283-287 AU - Morgan, K. E. AU - Zarembinski, T. I. AU - Theologis, A. AU - Abel, S. AU - VL - 454 UR - DO - 10.1016/S0014-5793(99)00819-4 AB - The plant hormone indoleacetic acid (IAA or auxin) transcriptionally activates a select set of early genes. The Auxl IAA class of early auxin-responsive genes encodes a large family of short-lived, nuclear proteins. Aux/IAA polypeptides homo-and heterodimerize, and interact with auxin-response transcription factors (ARFs) via C-terminal regions conserved in both protein families. This shared region contains a predicted βαα motif similar to the prokaryotic β-Ribbon DNA binding domain, which mediates both protein dimerization and DNA recognition. Here, we show by circular dichroism spectroscopy and by chemical cross-linking experiments that recombinant peptides corresponding to the predicted βαα region of three Aux/IAA proteins from Arabidopsis thaliana contain substantial α-helical secondary structure and undergo homo- and heterotypic interactions in vitro. Our results indicate a similar biochemical function of the plant βαα domain and suggest that the βαα fold plays an important role in mediating combinatorial interactions of Aux/IAA and ARF proteins to specifically regulate secondary gene expression in response to auxin. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2450 TI - Differential activation of the primary auxin response genes, PS-IAA4/5 and PS-IAA6, during early plant development JO - Plant J. PY - 1996 SP - 587-599 AU - Wong, L. M. AU - Abel, S. AU - Shen, N. AU - de la Foata, M. AU - Mall, Y. AU - Theologis, A. AU - VL - 9 UR - DO - 10.1046/j.1365-313X.1996.9050587.x AB - The plant growth hormone auxin typified by indoleacetic acid (IAA) transcriptionally activates early genes in pea, PS‐IAA4/5 and PS‐IAA6 , that are members of a multigene family encoding short‐lived nuclear proteins. To gain first insight into the biological role of PS‐IAA4/5 and PSIAA6 , promoter‐β‐glucuronidase (GUS) gene fusions were constructed and their expression during early development of transgenic tobacco seedlings was examined. The comparative analysis reveals spatial and temporal expression patterns of both genes that correlate with cells, tissues, and developmental processes known to be affected by auxin. GUS activity in seedlings of both transgenic lines is located in the root meristem, sites of lateral root initiation and in hypocotyls undergoing rapid elongation. In addition, mutually exclusive cell‐specific expression is evident. For instance, PS‐IAA4/5—GUS but not PS‐IAA6—GUS is expressed in root vascular tissue and in guard cells, whereas only PS‐IAA6—GUS activity is detectable in glandular trichomes and redistributes to the elongating side of the hypocotyl upon gravitropic stimulation. Expression of PS‐IAA4/5 and PS‐IAA6 in elongating, dividing, and differentiating cell types indicates multiple functions during development. The common and yet distinct activity patterns of both genes suggest a combinatorial code of spatio‐temporal co‐expression of the various PS‐IAA4/ 5‐like gene family members in plant development that may mediate cell‐specific responses to auxin. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2429 TI - Early Genes and Auxin Action JO - Plant Physiol. PY - 1996 SP - 9-17 AU - Abel, S. AU - Theologis, A. AU - VL - 111 UR - DO - 10.1104/pp.111.1.9 AB - A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2428 TI - DNA elements responsive to auxin JO - BioEssays PY - 1996 SP - 647-654 AU - Abel, S. AU - Ballas, N. AU - Wong, L.-M. AU - Theologis, A. AU - VL - 18 UR - DO - 10.1002/bies.950180808 AB - Genes induced by the plant hormone auxin are probably involved in the execution of vital cellular functions and developmental processes. Experimental approaches designed to elucidate the molecular mechanisms of auxin action have focused on auxin perception, genetic dissection of the signaling apparatus and specific gene activation. Auxin‐responsive promoter elements of early genes provide molecular tools for probing auxin signaling in reverse. Functional analysis of several auxin‐specific promoters of unrelated early genes suggests combinatorial utilization of both conserved and variable elements. These elements are arranged into autonomous domains and the combination of such modules generates uniquely composed promoters. Modular promoters allow for auxin‐mediated transcriptional responses to be revealed in a tissue‐ and development‐specific manner. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2452 TI - The PS-IAA4/5-like Family of Early Auxin-inducible mRNAs in Arabidopsis thaliana JO - J. Mol. Biol. PY - 1995 SP - 533-549 AU - Abel, S. AU - Nguyen, M. D. AU - Theologis, A. AU - VL - 251 UR - DO - 10.1006/jmbi.1995.0454 AB - The plant hormone auxin transcriptionally activates early genes. We have isolated a 14-member family of DNA sequences complementary to indoleacetic acid (IAA)-inducible transcripts inArabidopsis thaliana. The corresponding genes, IAA1 and IAA14, are homologs of PS-1AA4/5 and PS-IAA6 from pea, AUX22 and AUX28 from soybean, ARG3 and ARG4from mungbean, and AtAux2-11 and AtAux2-27 from Arabidopsis. The members of the family are differentially expressed in mature Arabidopsis plants. Characterization of IAA gene expression in etiolated seedlings demonstrates specificity for auxin inducibility. The response of most family members to IAA is rapid (within 4 to 30 minutes) and insensitive to cyclohexamide. Cyclohexamide alone induces all the early genes. Auxin-induction of two late genes, IAA7 and IAA8, is inhibited by cyclohexamide, indicating requirement of protein synthesis for their activation. All IAA genes display a biphasic dose response that is optimal at 10 μM IAA. However, individual genes respond differentially between 10 nM and 5μM IAA. Expression of all genes is defective in the Arabidopsis auxin-resistant mutant lines axr1, axr2, and aux1.The encoded polypeptides share four conserved domains, and seven invariant residues in the intervening regions. The spaces vary considerably in length, rendering the calculated molecular mass of IAA proteins to range from 19 kDa to 36 kDa. Overall sequence identity between members of the family is highly variable (36 to 87%). Their most significant structural features are functional nuclear transport signals, and a putative βαα-fold whose modeled three dimensional structure appears to be compatible with the prokaryotic β-ribbon DNA recognition motif. The data suggest that auxin induces in a differential and hierarchical fashion a large family of early genes that encode a structurally diverse class of nuclear proteins. These proteins are proposed to mediate tissue-specific and cell-type restricted responses to the hormone during plant growth and development. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2451 TI - A polymorphic bipartite motif signals nuclear targeting of early auxin-inducible proteins related to PS-IAA4 from pea (Pisum sativum) JO - Plant J. PY - 1995 SP - 87-96 AU - Abel, S. AU - Theologis, A. AU - VL - 8 UR - DO - 10.1046/j.1365-313X.1995.08010087.x AB - The plant hormone, indoleacetic acid (IAA), transcriptionally activates two early genes in pea, PS‐IAA4/5 and PS‐IAA6 , that encode short‐lived nuclear proteins. The identification of the nuclear localization signals (NLS) in PS‐IAA4 and PS‐IAA6 using progressive deletion analysis and site‐directed mutagenesis is reported. A C‐terminal SV40‐type NLS is sufficient to direct the β‐glucuronidase reporter to the nucleus of transiently transformed tobacco protoplasts, but is dispensible for nuclear localization of both proteins. The dominant and essential NLS in PS‐IAA4 and PS‐IAA6 overlap with a bipartite basic motif which is polymorphic and conserved in related proteins from other plant species, having the consensus sequence (KKNEK)KR‐X(24–71)‐(RSXRK)/(RK/RK). Both basic elements of this motif in PS‐IAA4, (KR‐X41‐RSYRK), function interdependently as a bipartite NLS. However, in PS‐IAA6 (KKNEKKR‐X36‐RKK) the upstream element of the corresponding motif contains additional basic residues which allow its autonomous function as an SV40‐type monopartite NLS. The spacer‐length polymorphism, X(24–70), in respective bipartite NLS peptides of several PS‐IAA4‐like proteins from Arabidopsis thaliana does not affect nuclear targeting function. The structural and functional variation of the bipartite basic motif in PS‐IAA4‐like proteins supports the proposed integrated consensus of NLS. A2 - C1 - Molecular Signal Processing ER - TY - JOUR ID - 2469 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 - 2468 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 -