TY - JOUR A1 - Zhang, Yunming A1 - Ramming, Anna A1 - Heinke, Lisa A1 - Altschmied, Lothar A1 - Slotkin, R. Keith A1 - Becker, Jörg D. A1 - Kappel, Christian A1 - Lenhard, Michael T1 - The poly(A) polymerase PAPS1 interacts with the RNA-directed DNA-methylation pathway in sporophyte and pollen development JF - The plant journal N2 - RNA-based processes play key roles in the regulation of eukaryotic gene expression. This includes both the processing of pre-mRNAs into mature mRNAs ready for translation and RNA-based silencing processes, such as RNA-directed DNA methylation (RdDM). Polyadenylation of pre-mRNAs is one important step in their processing and is carried out by three functionally specialized canonical nuclear poly(A) polymerases in Arabidopsis thaliana. Null mutations in one of these, termed PAPS1, result in a male gametophytic defect. Using a fluorescence-labelling strategy, we have characterized this defect in more detail using RNA and small-RNA sequencing. In addition to global defects in the expression of pollen-differentiation genes, paps1 null-mutant pollen shows a strong overaccumulation of transposable element (TE) transcripts, yet a depletion of 21- and particularly 24-nucleotide-long short interfering RNAs (siRNAs) and microRNAs (miRNAs) targeting the corresponding TEs. Double-mutant analyses support a specific functional interaction between PAPS1 and components of the RdDM pathway, as evident from strong synergistic phenotypes in mutant combinations involving paps1, but not paps2 paps4, mutations. In particular, the double-mutant of paps1 and rna-dependent rna polymerase 6 (rdr6) shows a synergistic developmental phenotype disrupting the formation of the transmitting tract in the female gynoecium. Thus, our findings in A. thaliana uncover a potentially general link between canonical poly(A) polymerases as components of mRNA processing and RdDM, reflecting an analogous interaction in fission yeast. KW - poly(A) polymerase KW - RNA-directed DNA methylation KW - pollen development KW - siRNAs KW - transposable elements KW - gynoecium development KW - Arabidopsis thaliana Y1 - 2019 U6 - https://doi.org/10.1111/tpj.14348 SN - 0960-7412 SN - 1365-313X VL - 99 IS - 4 SP - 655 EP - 672 PB - Wiley CY - Hoboken ER - TY - THES A1 - von Bismarck, Thekla T1 - The influence of long-term light acclimation on photosynthesis in dynamic light N2 - Photosynthesis converts light into metabolic energy which fuels plant growth. In nature, many factors influence light availability for photosynthesis on different time scales, from shading by leaves within seconds up to seasonal changes over months. Variability of light energy supply for photosynthesis can limit a plant´s biomass accumulation. Plants have evolved multiple strategies to cope with strongly fluctuation light (FL). These range from long-term optimization of leaf morphology and physiology and levels of pigments and proteins in a process called light acclimation, to rapid changes in protein activity within seconds. Therefore, uncovering how plants deal with FL on different time scales may provide key ideas for improving crop yield. Photosynthesis is not an isolated process but tightly integrates with metabolism through mutual regulatory interactions. We thus require mechanistic understanding of how long-term light acclimation shapes both, dynamic photosynthesis and its interactions with downstream metabolism. To approach this, we analyzed the influence of growth light on i) the function of known rapid photosynthesis regulators KEA3 and VCCN1 in dynamic photosynthesis (Chapter 2-3) and ii) the interconnection of photosynthesis with photorespiration (PR; Chapter 4). We approached topic (i) by quantifying the effect of different growth light regimes on photosynthesis and photoprotection by using kea3 and vccn1 mutants. Firstly, we found that, besides photosynthetic capacity, the activities of VCCN1 and KEA3 during a sudden high light phase also correlated with growth light intensity. This finding suggests regulation of both proteins by the capacity of downstream metabolism. Secondly, we showed that KEA3 accelerated photoprotective non-photochemical quenching (NPQ) kinetics in two ways: Directly via downregulating the lumen proton concentration and thereby de-activating pH-dependent NPQ, and indirectly via suppressing accumulation of the photoprotective pigment zeaxanthin. For topic (ii), we analyzed the role of PR, a process which recycles a toxic byproduct of the carbon fixation reactions, in metabolic flexibility in a dynamically changing light environment. For this we employed the mutants hpr1 and ggt1 with a partial block in PR. We characterized the function of PR during light acclimation by tracking molecular and physiological changes of the two mutants. Our data, in contrast to previous reports, disprove a generally stronger physiological relevance of PR under dynamic light conditions. Additionally, the two different mutants showed pronounced and distinct metabolic changes during acclimation to a condition inducing higher photosynthetic activity. This underlines that PR cannot be regarded purely as a cyclic detoxification pathway for 2PG. Instead, PR is highly interconnected with plant metabolism, with GGT1 and HPR1 representing distinct metabolic modulators. In summary, the presented work provides further insight into how energetic and metabolic flexibility is ensured by short-term regulators and PR during long-term light acclimation. N2 - Photosynthese wandelt Lichtenergie in metabolische Energie um, welche das Pflanzenwachstum antreibt. In der Natur wird die Verfügbarkeit von Licht von vielerlei Faktoren auf unterschiedlichen Zeitskalen beeinflusst, z. B. von der Beschattung durch Blätter innerhalb von Sekunden bis hin zu jahreszeitlichen Veränderungen über Monate. Fluktuationen in der Lichtenergieverfügbarkeit in der Natur kann die Biomasseakkumulation der Pflanzen limitieren. Pflanzen haben verschiedene Strategien entwickelt, um stark fluktuierendes Licht nutzen zu können. Diese reichen von der langfristigen Optimierung der Blattmorphologie und Physiologie und des Gehalts an Pigmenten und Proteinen in dem Prozess der Lichtakklimatisierung bis hin zu schnellen Veränderungen der Proteinaktivität innerhalb von Sekunden. Daher kann die Aufdeckung der Art und Weise, wie Pflanzen mit FL auf verschiedenen Zeitskalen umgehen, wichtige Ideen zur Verbesserung der Ernteerträge liefern. Die Photosynthese ist kein isolierter Prozess, sondern steht in enger Interaktion mit den nachgeschalteten Stoffwechselwegen. Daher benötigen wir mechanistisches Verständnis, wie Lichtakklimatisierung die dynamische Photosynthese als auch deren Interaktion mit Downstream-Metabolismus moduliert. Dafür haben wir den Einfluss von Lichtakklimatisierung auf i) die Funktion der schnellen Photosyntheseregulatoren KEA3 und VCCN1 in der dynamischen Photosynthese und ii) die flexible Interaktion von Photorespiration mit Photosynthese analysiert. Im ersten Themenkomplex (i) wurden die Auswirkungen verschiedener Wachstumslicht-bedingungen auf Photosynthese und Photoprotektion anhand von kea3- und vccn1-Mutanten quantifiziert. Zum einen konnten wir zeigen, dass neben der photosynthetischen Kapazität auch die Aktivitäten von VCCN1 und KEA3 während eines Hochlichtpulses mit der Wachstumslichtintensität korrelierten. Dies deutet auf eine Regulierung beider Proteine durch die Kapazität des Downstream-Metabolismus hin. Zum anderen beschleunigte KEA3 die Kinetik des photoprotektiven nicht-photochemischen Quenchings (NPQ) auf zweifache Weise: Direkt über die Herabregulierung der lumenalen Protonenkonzentration, was den pH-abhängigen NPQ deaktivierte, und indirekt über die Unterdrückung der Akkumulation des photoprotektiven Pigments Zeaxanthin. Für das zweite Thema (ii) untersuchten wir die Rolle des photorespiratorischen Metabolismus (PR), welcher ein toxisches Nebenprodukt der Kohlenstofffixierungsreaktionen recycelt, in der metabolischen Flexibilität in einer sich dynamisch verändernden Lichtumgebung. Dazu verwendeten wir die Mutanten hpr1 und ggt1 mit teilweise blockiertem PR Flux. Unsere Daten widerlegen, im Gegensatz zu früheren Berichten, eine allgemein größere physiologische Bedeutung von PR unter dynamischen Lichtbedingungen. Die beiden Mutanten zeigten ausgeprägte und distinkte metabolische Veränderungen während der Akklimatisierung an eine Bedingung mit höherer photosynthetischer Aktivität. Dies zeigt, dass PR nicht ausschließlich als zyklischer Entgiftungsweg für 2PG angesehen werden kann. Vielmehr ist PR tief in den pflanzlichen Stoffwechsel eingebettet, wobei GGT1 und HPR1 als distinkte Stellschrauben des Downstream-Metabolismus agieren. Zusammenfassend liefert die vorliegende Arbeit weitere Erkenntnisse darüber, wie die energetische und metabolische Flexibilität durch kurzfristige Regulatoren und den photorespiratorischen Metabolismus während der langfristigen Lichtakklimatisierung gewährleistet wird. KW - photosynthesis KW - fluctuating light KW - Arabidopsis thaliana KW - Photosynthese KW - fluktuierendes Licht Y1 - 2023 ER - TY - JOUR A1 - Lisso, Janina A1 - Altmann, Thomas A1 - Müssig, Carsten T1 - The AtNFXL1 gene encodes a NF-X1 type zinc finger protein required for growth under salt stress JF - FEBS letters : the journal for rapid publication of short reports in molecular biosciences N2 - The human NF-X1 protein and homologous proteins in eukaryotes represent a class of transcription factors which are characterised. by NF-X1 type zinc finger motifs. The Arabidopsis genome encodes two NF-X1 homologs, which we termed AtNFXL1 and AtNFXL2. Growth and survival was impaired in atnfxl1 knock-out mutants and AtNFXL1-antisense plants under salt stress in comparison to wild-type plants. In contrast, 35S: :AtNFXL1 plants showed higher survival rates. The AtNFXL2 protein potentially plays an antagonistic role. The Arabidopsis NF-X1 type zinc finger proteins likely are part of regulatory mechanisms, which protect major processes such as photosynthesis. KW - Arabidopsis thaliana KW - NF-X1 KW - salt stress Y1 - 2006 U6 - https://doi.org/10.1016/j.febslet.2006.07.079 SN - 0014-5793 VL - 580 IS - 22 SP - 4851 EP - 4856 PB - Elsevier CY - Amsterdam ER - TY - JOUR A1 - Liu, Qingting A1 - Li, Xiaoping A1 - Fettke, Jörg T1 - Starch granules in Arabidopsis thaliana mesophyll and guard cells show similar morphology but differences in size and number JF - International journal of molecular sciences N2 - Transitory starch granules result from complex carbon turnover and display specific situations during starch synthesis and degradation. The fundamental mechanisms that specify starch granule characteristics, such as granule size, morphology, and the number per chloroplast, are largely unknown. However, transitory starch is found in the various cells of the leaves of Arabidopsis thaliana, but comparative analyses are lacking. Here, we adopted a fast method of laser confocal scanning microscopy to analyze the starch granules in a series of Arabidopsis mutants with altered starch metabolism. This allowed us to separately analyze the starch particles in the mesophyll and in guard cells. In all mutants, the guard cells were always found to contain more but smaller plastidial starch granules than mesophyll cells. The morphological properties of the starch granules, however, were indiscernible or identical in both types of leaf cells. KW - starch granules KW - starch granule number per chloroplast KW - starch morphology KW - mesophyll cell KW - guard cell KW - LCSM KW - Arabidopsis thaliana KW - starch granule initiation KW - starch metabolism Y1 - 2021 U6 - https://doi.org/10.3390/ijms22115666 SN - 1422-0067 SN - 1661-6596 VL - 22 IS - 11 PB - Molecular Diversity Preservation International CY - Basel ER - TY - GEN A1 - Liu, Qingting A1 - Li, Xiaoping A1 - Fettke, Jörg T1 - Starch granules in Arabidopsis thaliana mesophyll and guard cells show similar morphology but differences in size and number T2 - Postprints der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe N2 - Transitory starch granules result from complex carbon turnover and display specific situations during starch synthesis and degradation. The fundamental mechanisms that specify starch granule characteristics, such as granule size, morphology, and the number per chloroplast, are largely unknown. However, transitory starch is found in the various cells of the leaves of Arabidopsis thaliana, but comparative analyses are lacking. Here, we adopted a fast method of laser confocal scanning microscopy to analyze the starch granules in a series of Arabidopsis mutants with altered starch metabolism. This allowed us to separately analyze the starch particles in the mesophyll and in guard cells. In all mutants, the guard cells were always found to contain more but smaller plastidial starch granules than mesophyll cells. The morphological properties of the starch granules, however, were indiscernible or identical in both types of leaf cells. T3 - Zweitveröffentlichungen der Universität Potsdam : Mathematisch-Naturwissenschaftliche Reihe - 1143 KW - starch granules KW - starch metabolism KW - starch granule initiation KW - starch granule number per chloroplast KW - starch morphology KW - mesophyll cell KW - guard cell KW - LCSM KW - Arabidopsis thaliana Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-511067 SN - 1866-8372 IS - 1143 ER - TY - JOUR A1 - Merida, Angel A1 - Fettke, Jörg T1 - Starch granule initiation in Arabidopsis thaliana chloroplasts JF - The plant journal N2 - The initiation of starch granule formation and the mechanism controlling the number of granules per plastid have been some of the most elusive aspects of starch metabolism. This review covers the advances made in the study of these processes. The analyses presented herein depict a scenario in which starch synthase isoform 4 (SS4) provides the elongating activity necessary for the initiation of starch granule formation. However, this protein does not act alone; other polypeptides are required for the initiation of an appropriate number of starch granules per chloroplast. The functions of this group of polypeptides include providing suitable substrates (maltooligosaccharides) to SS4, the localization of the starch initiation machinery to the thylakoid membranes, and facilitating the correct folding of SS4. The number of starch granules per chloroplast is tightly regulated and depends on the developmental stage of the leaves and their metabolic status. Plastidial phosphorylase (PHS1) and other enzymes play an essential role in this process since they are necessary for the synthesis of the substrates used by the initiation machinery. The mechanism of starch granule formation initiation in Arabidopsis seems to be generalizable to other plants and also to the synthesis of long-term storage starch. The latter, however, shows specific features due to the presence of more isoforms, the absence of constantly recurring starch synthesis and degradation, and the metabolic characteristics of the storage sink organs. KW - starch granules KW - starch metabolism KW - starch granule initiation KW - starch KW - granule number per chloroplast KW - starch morphology KW - Arabidopsis thaliana Y1 - 2021 U6 - https://doi.org/10.1111/tpj.15359 SN - 0960-7412 SN - 1365-313X VL - 107 IS - 3 SP - 688 EP - 697 PB - Wiley CY - Hoboken ER - TY - THES A1 - Martinez-Seidel, Federico T1 - Ribosome Heterogeneity and Specialization during Temperature Acclimation in Plants N2 - Ribosomes decode mRNA to synthesize proteins. Ribosomes, once considered static, executing machines, are now viewed as dynamic modulators of translation. Increasingly detailed analyses of structural ribosome heterogeneity led to a paradigm shift toward ribosome specialization for selective translation. As sessile organisms, plants cannot escape harmful environments and evolved strategies to withstand. Plant cytosolic ribosomes are in some respects more diverse than those of other metazoans. This diversity may contribute to plant stress acclimation. The goal of this thesis was to determine whether plants use ribosome heterogeneity to regulate protein synthesis through specialized translation. I focused on temperature acclimation, specifically on shifts to low temperatures. During cold acclimation, Arabidopsis ceases growth for seven days while establishing the responses required to resume growth. Earlier results indicate that ribosome biogenesis is essential for cold acclimation. REIL mutants (reil-dkos) lacking a 60S maturation factor do not acclimate successfully and do not resume growth. Using these genotypes, I ascribed cold-induced defects of ribosome biogenesis to the assembly of the polypeptide exit tunnel (PET) by performing spatial statistics of rProtein changes mapped onto the plant 80S structure. I discovered that growth cessation and PET remodeling also occurs in barley, suggesting a general cold response in plants. Cold triggered PET remodeling is consistent with the function of Rei-1, a REIL homolog of yeast, which performs PET quality control. Using seminal data of ribosome specialization, I show that yeast remodels the tRNA entry site of ribosomes upon change of carbon sources and demonstrate that spatially constrained remodeling of ribosomes in metazoans may modulate protein synthesis. I argue that regional remodeling may be a form of ribosome specialization and show that heterogeneous cytosolic polysomes accumulate after cold acclimation, leading to shifts in the translational output that differs between wild-type and reil-dkos. I found that heterogeneous complexes consist of newly synthesized and reused proteins. I propose that tailored ribosome complexes enable free 60S subunits to select specific 48S initiation complexes for translation. Cold acclimated ribosomes through ribosome remodeling synthesize a novel proteome consistent with known mechanisms of cold acclimation. The main hypothesis arising from my thesis is that heterogeneous/ specialized ribosomes alter translation preferences, adjust the proteome and thereby activate plant programs for successful cold acclimation. N2 - Ribosomen dekodieren mRNA, um Proteine zu synthetisieren. Ribosomen, früher als statische, ausführende Maschinen betrachtet, werden heute als dynamische Modulatoren der Translation angesehen. Zunehmend detailliertere Analysen der Strukturheterogenität von Ribosomen führte zu einem Paradigmenwechsel hin zu einer Spezialisierung von Ribosomen für eine selektive Translation. Als sessile Organismen können Pflanzen schädlichen Umwelteinflüssen nicht ausweichen und haben Strategien entwickelt, um diesen zu widerstehen. Zytosolische Ribosomen von Pflanzen sind in mancher Hinsicht vielfältiger, als die von anderen Metazoen. Diese Vielfalt könnte zur Stressakklimatisierung der Pflanzen beitragen. Ziel dieser Arbeit war es, festzustellen, ob Pflanzen die Heterogenität der Ribosomen nutzen, um die Proteinsynthese durch spezialisierte Translation zu regulieren. Ich habe mich auf die Temperaturakklimatisierung konzentriert, insbesondere auf den Wechsel zu niedrigen Temperaturen. Im Verlauf der Kälteakklimatisierung stellt Arabidopsis das Wachstum für sieben Tage ein. Währenddessen etabliert sie die für die Wiederaufnahme des Wachstums erforderlichen Anpassungen. Vorherige Ergebnisse deuten darauf hin, dass Ribosomenbiogenese für die Kälteakklimatisierung essentiell ist. REIL-Mutanten (reil-dkos), denen ein 60S-Reifungsfaktor fehlt, akklimatisieren sich nicht erfolgreich und nehmen das Wachstum nicht wieder auf. Anhand dieser Genotypen habe ich kältebedingte Defekte der Ribosomenbiogenese auf den Aufbau des Polypeptidaustritts-Tunnels (PET) zurückgeführt, indem ich räumliche statistische Analysen von rProtein-Veränderungen auf die pflanzliche 80S-Struktur abgebildet habe. Ich habe entdeckt, dass Wachstumsstillstand und PET-Umbau auch in Gerste auftreten, was auf eine allgemeine Kältereaktion in Pflanzen hindeutet. Der durch Kälte ausgelöste PET-Umbau stimmt über ein mit der Funktion von Rei-1, einem REIL-homologen Protein aus Hefe, in der Rei-1 die PET-Qualitätskontrolle durchführt. Anhand bahnbrechender Daten zur Ribosomenspezialisierung zeige ich, dass Hefe die tRNA-Eintrittsstelle von Ribosomen bei einem Wechsel von Kohlenstoffquellen umbaut, und demonstriere, dass ein räumlich begrenzter Umbau von Ribosomen in Metazoen die Proteinsynthese modulieren kann. Ich argumentiere, dass die regionale Umgestaltung eine Form der Ribosomenspezialisierung sein kann, und zeige, dass nach einer Kälteakklimatisierung heterogene zytosolische Polysomen akkumulieren, was zu Verschiebungen im Translationsoutput führt, der sich zwischen Wildtyp und reil-dkos unterscheidet. Ich habe festgestellt, dass die heterogenen Komplexe aus neu synthetisierten und wiederverwendeten Proteinen bestehen. Ich schlage vor, dass maßgeschneiderte Ribosomenkomplexe freie 60S-Untereinheiten in die Lage versetzen, spezifische 48S-Initiationskomplexe für die Translation auszuwählen. Kälte-akklimatisierte Ribosomen synthetisieren durch Ribosomenumbau ein neues Proteom, das mit bekannten Mechanismen der Kälteakklimatisierung übereinstimmt. Die Haupthypothese, die sich aus meiner Arbeit ergibt, ist, dass heterogene/spezialisierte Ribosomen ihre Translationspräferenzen verändern, das Proteom anpassen und dadurch Pflanzenprogramme für eine erfolgreiche Kälteakklimatisierung aktivieren. T2 - Ribosomenheterogenität und -spezialisierung während der Temperaturakklimatisierung in Pflanzen KW - Ribosome specialization KW - Ribosomal protein heterogeneity KW - Ribosomal protein substoichiometry KW - Protein synthesis KW - Translational regulation KW - Plant cytosolic translation KW - Cold acclimation KW - Ribosome biogenesis KW - 60S maturation KW - Hordeum vulgare KW - Arabidopsis thaliana KW - 60S-Reifung KW - Kälteakklimatisierung KW - Cytosolische Translation in Pflanzen KW - Proteinsynthese KW - Ribosomale Proteinheterogenität KW - Ribosomale Protein Substöchiometrie KW - Ribosomen-Biogenese KW - Ribosomen-Spezialisierung KW - Translationsregulation Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus4-580724 ER - TY - JOUR A1 - Pandey, Prashant K. A1 - Yu, Jing A1 - Omranian, Nooshin A1 - Alseekh, Saleh A1 - Vaid, Neha A1 - Fernie, Alisdair R. A1 - Nikoloski, Zoran A1 - Laitinen, Roosa A. E. T1 - Plasticity in metabolism underpins local responses to nitrogen in Arabidopsis thaliana populations JF - Plant Direct N2 - Nitrogen (N) is central for plant growth, and metabolic plasticity can provide a strategy to respond to changing N availability. We showed that two local A. thaliana populations exhibited differential plasticity in the compounds of photorespiratory and starch degradation pathways in response to three N conditions. Association of metabolite levels with growth-related and fitness traits indicated that controlled plasticity in these pathways could contribute to local adaptation and play a role in plant evolution. KW - Arabidopsis thaliana KW - natural variation KW - nitrogen availability KW - photorespiration KW - plasticity Y1 - 2019 U6 - https://doi.org/10.1002/pld3.186 SN - 2475-4455 VL - 3 IS - 11 PB - John Wiley & sonst LTD CY - Chichester ER - TY - JOUR A1 - Ralevski, Alexandra A1 - Apelt, Federico A1 - Olas, Justyna Jadwiga A1 - Müller-Röber, Bernd A1 - Rugarli, Elena I. A1 - Kragler, Friedrich A1 - Horvath, Tamas L. T1 - Plant mitochondrial FMT and its mammalian homolog CLUH controls development and behavior in Arabidopsis and locomotion in mice JF - Cellular and molecular life sciences N2 - Mitochondria in animals are associated with development, as well as physiological and pathological behaviors. Several conserved mitochondrial genes exist between plants and higher eukaryotes. Yet, the similarities in mitochondrial function between plant and animal species is poorly understood. Here, we show that FMT (FRIENDLY MITOCHONDRIA) from Arabidopsis thaliana, a highly conserved homolog of the mammalian CLUH (CLUSTERED MITOCHONDRIA) gene family encoding mitochondrial proteins associated with developmental alterations and adult physiological and pathological behaviors, affects whole plant morphology and development under both stressed and normal growth conditions. FMT was found to regulate mitochondrial morphology and dynamics, germination, and flowering time. It also affects leaf expansion growth, salt stress responses and hyponastic behavior, including changes in speed of hyponastic movements. Strikingly, Cluh(+/-) heterozygous knockout mice also displayed altered locomotive movements, traveling for shorter distances and had slower average and maximum speeds in the open field test. These observations indicate that homologous mitochondrial genes may play similar roles and affect homologous functions in both plants and animals. KW - Arabidopsis thaliana KW - Mitochondria KW - FMT KW - Hyponasty KW - Mice KW - CLUH; KW - Locomotion Y1 - 2022 U6 - https://doi.org/10.1007/s00018-022-04382-3 SN - 1420-682X SN - 1420-9071 VL - 79 IS - 6 PB - Springer International Publishing AG CY - Cham (ZG) ER - TY - THES A1 - Nikolovski, Nino T1 - Pectin: New insights from an old polymer through pectinase-based genetic screens T1 - Pektin: Neue Einblicke in ein altes Polymer durch Pektinase-basierte genetische Screens N2 - Pectic polysaccharides, a class of plant cell wall polymers, form one of the most complex networks known in nature. Despite their complex structure and their importance in plant biology, little is known about the molecular mechanism of their biosynthesis, modification, and turnover, particularly their structure-function relationship. One way to gain insight into pectin metabolism is the identification of mutants with an altered pectin structure. Those were obtained by a recently developed pectinase-based genetic screen. Arabidopsis thaliana seedlings grown in liquid medium containing pectinase solutions exhibited particular phenotypes: they were dwarfed and slightly chlorotic. However, when genetically different A. thaliana seed populations (random T-DNA insertional populations as well as EMS-mutagenized populations and natural variations) were subjected to this treatment, individuals were identified that exhibit a different visible phenotype compared to wild type or other ecotypes and may thus contain a different pectin structure (pec-mutants). After confirming that the altered phenotype occurs only when the pectinase is present, the EMS mutants were subjected to a detailed cell wall analysis with particular emphasis on pectins. This suite of mutants identified in this study is a valuable resource for further analysis on how the pectin network is regulated, synthesized and modified. Flanking sequences of some of the T-DNA lines have pointed toward several interesting genes, one of which is PEC100. This gene encodes a putative sugar transporter gene, which, based on our data, is implicated in rhamnogalacturonan-I synthesis. The subcellular localization of PEC100 was studied by GFP fusion and this protein was found to be localized to the Golgi apparatus, the organelle where pectin biosynthesis occurs. Arabidopsis ecotype C24 was identified as a susceptible one when grown with pectinases in liquid culture and had a different oligogalacturonide mass profile when compared to ecotype Col-0. Pectic oligosaccharides have been postulated to be signal molecules involved in plant pathogen defense mechanisms. Indeed, C24 showed elevated accumulation of reactive oxygen species upon pectinase elicitation and had altered response to the pathogen Alternaria brassicicola in comparison to Col-0. Using a recombinant inbred line population three major QTLs were identified to be responsible for the susceptibility of C24 to pectinases. In a reverse genetic approach members of the qua2 (putative pectin methyltransferase) family were tested for potential target genes that affect pectin methyl-esterification. The list of these genes was determined by in silico study of the pattern of expression and co-expression of all 34 members of this family resulting in 6 candidate genes. For only for one of the 6 analyzed genes a difference in the oligogalacturonide mass profile was observed in the corresponding knock-out lines, confirming the hypothesis that the methyl-esterification pattern of pectin is fine tuned by members of this gene family. This study of pectic polysaccharides through forward and reverse genetic screens gave new insight into how pectin structure is regulated and modified, and how these modifications could influence pectin mediated signalling and pathogenicity. N2 - Pektin Polysaccharide, eine Klasse pflanzlicher Zellwand Polymere, formen eine der komplexesten natürlichen Strukturen. Trotz seiner immensen Bedeutung in der Biologie der Pflanzen sind die Kenntisse über die molekularen Mechanismen der Pektin Biosynthese, dessen Modifikation und Abbau überraschend gering. Eine Möglichkeit neue Einblicke in den pflanzlichen Pektin Metabolismus zu erhalten, ist die Identifizierung von Mutanten mit veränderter Pektinstruktur. Solche Mutanten konnten durch ein neuatiges Selektionsverfahren gefunden werden. Zieht man Keimlinge der Ackerschmalwand (Arabidopsis thaliana) in Flüssigmedium mit Pektinase an, so lässt sich ein typischer Phänotyp beobachten: Die Pflanzen sind kleinwüchsig und leicht chlorotisch. Diesem Verfahren wurden Populationen verschiedener Genotypen (Insertions Linien, EMS Mutanten, natürlich vorkommende Varianten) ausgesetzt. Auf diese Weise wurden Individuen identifiziert, die gegenüber der Pektinase Behandlung eine verminderte oder erhöhte Resistenz aufweisen, was auf eine veränderte Pektinstruktur hindeutet. Die EMS Mutanten wurden einer detaillierten Zellwand Analyse unterzogen. die so in dieser Arbeit identifizierte Kollektion von Mutanten stellt eine wertvolle Ressource für weitere Forschungsansätze zur Regulation, Biosynthese und Modifikation des Pektins dar. Die Lokalisation der Insertionen in den T-DNA Linien führte zur Identifikation interessanter Gene, zu denen der putative Zuckertransporter PEC100 gehört. Dieses Gen steht vermutlich in Verbindung mit der Synthese von Rhamnogalakturonan-I, einem Bestandteil des Pektins. In dieser Arbeit konnte PEC100 im Golgi Apparat, dem Ort der Pektin Biosynthese, lokalisiert werden. Die natürlich vorkommende Variante C24 ist besonders empfindlich gegenüber der Pektinase. Diese Empfindlichkeit konnte anhand rekombinanter Inzucht Linien auf drei bedeutende quantitative Merkmalsloci (QTL) eingegrenzt werden. C24 zeigte zudem ein gegenüber der Referenz verändertes Massenprofil der Oligogalakturonide. Diese werden derzeit als Signalmoleküle in der pflanzlichen Pathogenabwehr diskutiert, was mit der in dieser Arbeit geseigten Resistenz von C24 gegenüber Schwarzfleckigkeit verursachende Pilz (Alternaria brassicicola) korreliert. In einem revers-genetischen Ansatz wurden zudem Mitglieder der Pektin Methyltransferase Familie als potentielle Enzyme getestet, die die Pektin Methylesterifikation beeinflussen könnten. Diese Mutation in einer dieser Methyltransferasen führte zu Veränderungen des Oligogalakturonid Massenprofils. Dies bestätigt die Hypothese, dass Mitglieder dieser Genfamilie an der Regulation der Methylesterifikation von Pektin beteiligt sind. Die vorliegende Studie, in der ein genetishen Selektionverfahren und Methoden der reversen Genetik kombiniert wurden, hat neue Einblicke in die Regulation und Modifikation von Pektin geliefert. KW - Pektin KW - Pektinase KW - genetischer Screen KW - Arabidopsis thaliana KW - Zellwand KW - pectin KW - pectinase KW - genetic screen KW - Arabidopsis thaliana KW - cell wall Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-opus-35255 ER -