Molekulare, physiologische und biochemische Charakterisierung der Genfamilie für 4-Cumarat:Coenzym A Ligase aus Sojabohne (Glycine max L.)

Die 4-Cumarat:Coenzym A Ligase (4CL) ist ein Enzym des allgemeinen Phenylpropanstoffwechsels, das aktivierte Hydroxyzimtsäure-Ester zur Synthese spezifischer Phenylpropanoide bereitstellt. In vielen Pflanzen kommen strukturell und funktionell sehr ähnliche 4CL-Isoformen vor, weshalb diesem Enzym dort nur eine beschränkte regulatorische Funktion bei der Verteilung der unterschiedlich substituierten Zimtsäurederivate für nachfolgende Synthesen zugesprochen wird. Durch Isolierung der cDNA der von Knobloch und Hahlbrock (1975) partiell gereinigten Ligase 1 und der Vervollständigung der kodierenden Sequenz der Gm4CL4 konnte mit großer Wahrscheinlichkeit die Genfamilie der Sojabohne-4CLs komplettiert werden. Mit der cDNA der Gm4CL1 war außerdem erstmals eine 4CL-Sequenz verfügbar, die für eine Sinapinsäure-umsetzende 4CL kodiert. Zusammen mit den bereits beschriebenen 4CL-Isoenzymen 2 und 3 (Uhlmann und Ebel, 1993; Möllers, 1997) existieren in Sojabohne somit mindestens vier 4CL-Isoformen. Die unterschiedlichen Substratspezifitäten der rekombinanten Proteine, die Fähigkeit, die gesamte Gruppe der pflanzlichen Hxdroxyzimtsäuren zu aktivieren und die differentielle Synthese der Gm4CL-Isoformen nach Elicitor-Behandlung von Sojabohnezellkulturen bzw. Infektion von Sojabohnekeimlingen weisen auf eine bedeutende regulatorische Funktion der 4CL hinsichtlich der Bereitstellung unterschiedlich substituierter Zimtsäurederivate zur Synthese spezifische Phenylpropanoide in Sojabohne hin. Dabei scheinen Gm4CL1 und Gm4CL2 vor allem an der Synthese von Phenylpropanoiden beteiligt zu sein, die für Wachstum und Entwicklung der Pflanzen benötigt werden, während Gm4CL3 und Gm4CL4 aktivierte Zimtsäuren bereitstellen, die aufgrund von Umweltveränderungen benötigt werden. Die 4CL wird mit Acyl-CoA Ligasen, Peptidsynthetasen und Luciferase zur Familie der AMP-bindenden Proteine zusammengefaßt. Diesen Enzymen ist nicht nur der Mechanismus der AMP-Aktivierung gemeinsam, sondern sie besitzen auch Ähnlichkeiten in ihren Proteinstrukturen. Durch Bildung von Hybridenzymen zwischen Gm4CL1 und Gm4CL3 konnte der zentrale Sequenzbereich der 4CL als Substratspezifität-determinierende Region ermittelt werden. Mit Hilfe der Informationen, die von der Kristallstruktur der Phenylalaninaktivierenden Untereinheit der Gramicidin-S-Synthetase gewonnen wurden, konnten vier mögliche Substratbindemotive der 4CL identifiziert werden. Innerhalb eines dieser Motive unterscheidet sich die Gm4CL1 von allen anderen bisher klonierten 4CLs durch den Verlust eines Aminosäurerestes, wodurch die Gm4CL1 in der Lage ist, hochsubstituierte Zimsäurederivate zu aktivieren. Wird der entsprechende Aminosäurerest der Gm4CL2 (Gm4CL2dV345) und Gm4CL3 (Gm4CL3dV367) entfernt, können beide Deletionsmutanten Sinapinsäure umsetzen. Außerdem ist Gm4CL3dV367 in der Lage, 3,4-Dimethoxyzimtsäure zu aktivieren und zeigt eine deutlich erhöhte Affinität gegenüber Ferulasäure. Durch das Entfernen einer einzigen Aminosäure können somit hochsubstituierte Zimtsäurederivate umgesetzt werden. Die Möglichkeit, die Substratspezifität dieses zentralen Enzymes des Phenylpropanstoffwechsels zu modifizieren, eröffnet die Herstellung transgener Pflanzen mit gewünschtem Phenylpropanoid-Profil

The role of nitric oxide in plant biology:Current insights and future perspectives

Nitric oxide (NO) is a redox-active gaseous signal uniformly present in eukaryotes, but its formation, signalling, and effects are specific within the plant kingdom in several aspects. NO synthesis in algae proceeds by mechanisms similar to that in mammals, but there are different pathways in higher plants. Beyond synthesis, the regulatory processes to maintain steady-state NO levels are also integral for the projection of NO function. As a key redox molecule, NO exhibits a number of pivotal molecular interactions, for example with reactive oxygen species, hydrogen sulfide, and calcium, with these molecular interplays largely underpinning NO bioactivity. In this context, NO has emerged as a key regulator in plant growth, development, and environmental interactions. In this special issue, a collection of reviews discusses the current state-of-the-art and possible future directions related to the biology and chemistry of plant NO function

Effect of nitric oxide on gene transcription – S-nitrosylation of nuclear proteins

Nitric oxide (NO) plays an important role in many different physiological processes in plants. It mainly acts by post-translationally modifying proteins. Modification of cysteine residues termed as S-nitrosylation is believed to be the most important mechanism for transduction of bioactivity of NO. The first proteins found to be nitrosylated were mainly of cytoplasmic origin or isolated from mitochondria and peroxisomes. Interestingly, it was shown that redox-sensitive transcription factors are also nitrosylated and that NO influences the redox-dependent nuclear transport of some proteins. This implies that NO plays a role in regulating transcription and/or general nuclear metabolism which is a fascinating new aspect of NO signaling in plants. In this review, we will discuss the impact of S-nitrosylation on nuclear plant proteins with a focus on transcriptional regulation, describe the function of this modification and draw also comparisons to the animal system in which S-nitrosylation of nuclear proteins is a well characterized concept

Editorial: Post-translational Modifications in Plant Nuclear Signaling: Novel Insights Into Responses to Environmental Changes

Just imagine a Plant Science professor in front of a classroom full of interested and attentive students. Imagine what their answers to this intriguing question would be: “What are, according to you, the functions ensured by the plant cell nucleus?” It would be very surprising if some of them would answer cell signaling in response to biotic and abiotic stresses or developmental processes. Most of them would probably answer according to a classical point of view: DNA replication or gene expression. Hence it is still admitted in recent publications (see for instance Fedorenko et al., 2010) that molecules smaller than 40 kDa can diffuse freely across the nuclear envelope pores. However, Pauly et al. (2000) showed by studying nuclear Ca2+ signaling that elevations in the extranuclear Ca2+ concentration do not induce an automatic increase of nuclear [Ca2+] as it could be expected. Hence Ca2+ does not freely diffuse across the nuclear envelop pores, indicating that its transport is finely regulated. Then it becomes evident that we should now consider the nucleus as a key component of cell signaling processes leading to the regulation of specific sets of genes. The aim of this topic was to point out how nuclear post-translational modifications (PTMs) play fundamental roles in the signaling pathways initiated in response to environmental changes. Two major points were assessed. First, different papers clearly demonstrate that the nucleus is fully equipped to perform the main PTMs: (de)phosphorylation (Bigeard and Hirt; Krysan and Colcombet), (de)acetylation (Luo et al.; Fül et al.; Ramirez-Prado et al.), oxidoreduction (Martins et al.) or SUMOylation/ubiquitination (Mazur et al.; Serrano et al.). Hence the nucleus can easily integrate a complex network of second messengers including changes in Ca2+ concentration, reactive oxygen species or nitric oxide. Martins et al. nicely exemplified how changes in the nuclear redox status regulate fundamental processes such as cell cycle, protein transport or transcription via S-nitrosylation or S-glutationylation. Activation of nuclear PTMs can also be achieved by the translocation of enzymes which are both substrates and effectors of these PTMs. This is the case for mitogen-activated protein kinases (MAPKs) that in some specific contexts, translocate from the cytosol to the nucleus upon their activation by their corresponding MAPK kinases (Bigeard and Hirt). Relocation of proteins in response to or through PTMs can also be considered at the intranuclear level. Hence in response to SUMO conjugation several Arabidopsis transcription factors were shown to be re-localized in certain nuclear foci (Mazur et al.). PTMs can also affect the behavior of nuclear proteins and in fine their activity. Serrano et al. highlight how the ubiquitin-proteasome system contributes to the nuclear proteome plasticity. Focusing on E3-Ub-ligases, they illustrate how these enzymes attenuate the signaling pathway once the stress has ceased and how they control the homeostasis of nuclear proteins (transcription factors, immune receptors). The second major point of this topic concerns the target proteins of these PTMs. Of course histones are a piece of choice. The review by Ramirez-Prado et al. illustrates how removing or adding marks (phosphorylation, acetylation, methylation, or ubiquitination) on specific histone lysine residues associated to defense genes (WRKYs, pathogenesis-related proteins, etc.) mostly under the control of salicylic acid or jasmonic acid/ethylene signaling pathways, will control the outcome of the plant-microorganism interaction. A second interesting aspect in this review is the illustration of how pathogens manipulate the chromatin regulatory network of the host to achieve their infection process through for example the production of toxins inhibiting histone deacetylases (HDACs), leading to plant susceptibility. PTMs on histones are also of major importance in the response to abiotic stresses. Luo et al reviewed how HDACs, by deacetylating specific lysine residues (mainly H3K9, H3K14, and H4K15) of specific genes regulate responses to salt, drought, cold or heat. However, chromatin remodeling and in fine regulation of gene expression is not only linked to histone modifications. Fül et al. as a perspective in this topic remind us that subunits of key chromatin remodelers (such as MED12, MED13, and MED19A) or transcription factors like Yin Yang 1 in the response to abscisic acid are also targeted and regulated by lysine deacetylases. Hence lysine (de)acetylation regulates gene expression by acting on histones, but also on the whole transcriptional machinery. In conclusion this topic clearly illustrates the diversity of nuclear PTMs, the crosstalks between some of them, and finally their major roles in the regulation of gene expression. A first fascinating challenge is now to decipher at the molecular level the ways cells are using to translocate signals from cytoplasm to the nucleus that regulate nuclear PTMs. A second one is to go further in the characterization of their target proteins: as it is now nicely exemplified by the interplay of histone marks in epigenetics, this is the sine qua non condition to fully understand the power of nuclear PTMs

Deciphering the S-Nitrosylome of Arabipdosis thaliana during the defense response

Comunicaciones a congreso

Proteomic identification of s-nitrosylated proteins in arabidopsis thaliana in response to pathogen infection

Comunicaciones a congreso

Ethylene supports colonization of plant roots by the mutualistic fungus Piriformospora indica

The mutualistic basidiomycete Piriformospora indica colonizes roots of mono- and dicotyledonous plants, and thereby improves plant health and yield. Given the capability of P. indica to colonize a broad range of hosts, it must be anticipated that the fungus has evolved efficient strategies to overcome plant immunity and to establish a proper environment for nutrient acquisition and reproduction. Global gene expression studies in barley identified various ethylene synthesis and signaling components that were differentially regulated in P. indica-colonized roots. Based on these findings we examined the impact of ethylene in the symbiotic association. The data presented here suggest that P. indica induces ethylene synthesis in barley and Arabidopsis roots during colonization. Moreover, impaired ethylene signaling resulted in reduced root colonization, Arabidopsis mutants exhibiting constitutive ethylene signaling, -synthesis or ethylene-related defense were hyper-susceptible to P. indica. Our data suggest that ethylene signaling is required for symbiotic root colonization by P. indica

Computational prediction of NO-dependent posttranslational modifications in plants: Current status and perspectives

The perception and transduction of nitric oxide (NO) signal is achieved by NO-dependent posttranslational modifications (PTMs) among which S-nitrosation and tyrosine nitration has biological significance. In plants, 100-1000 S-nitrosated and tyrosine nitrated proteins have been identified so far by mass spectrometry. The determination of NO-modified protein targets/amino acid residues is often methodologically challenging. In the past decade, the growing demand for the knowledge of S-nitrosated or tyrosine nitrated sites has motivated the introduction of bioinformatics tools. For predicting S-nitrosation seven computational tools have been developed (GPS-SNO, SNOSite, iSNO-PseACC, iSNO-AAPAir, PSNO, PreSNO, RecSNO). Four predictors have been developed for indicating tyrosine nitration sites (GPS-YNO2, iNitro-Tyr, PredNTS, iNitroY-Deep), and one tool (DeepNitro) predicts both NO-dependent PTMs. The advantage of these computational tools is the fast provision of large amount of information. In this review, the available software tools have been tested on plant proteins in which S-nitrosated or tyrosine nitrated sites have been experimentally identified. The predictors showed distinct performance and there were differences from the experimental results partly due to the fact that the three-dimensional protein structure is not taken into account by the computational tools. Nevertheless, the predictors excellently establish experiments, and it is suggested to apply all available tools on target proteins and compare their results. In the future, computational prediction must be developed further to improve the precision with which S-nitrosation/tyrosine nitration-sites are identified