Fakultät Naturwissenschaften

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Biologie, Ernährungs-wissenschaften und Lebensmittelwissenschaften sind die Schwerpunkte der Fakultät. Die Forschung befasst sich mit Schlüsselthemen der Life Sciences.

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Bioactive compounds and antioxidant activity in the fruit of rosehip (Rosa canina L. and Rosa rubiginosa L.)
(2023) Peña, Fabiola; Valencia, Sebastián; Tereucán, Gonzalo; Nahuelcura, Javiera; Jiménez-Aspee, Felipe; Cornejo, Pablo; Ruiz, Antonieta
Rosehips (Rosa spp., Rosaceae) are wild rose bushes with more than 100 species. Its fruits vary in colour and size, depending on the species, and are recognised for their nutritional characteristics. Ten samples of Rosa canina L. and Rosa rubiginosa L. fruits were collected at different geographical points from Southern Chile. Nutrients such as crude protein and minerals and functional properties such as phenolic compounds, ascorbic acid, and also antioxidant activities were evaluated by HPLC-DAD-ESI-MS/MS. The results revealed a high content of bioactive compounds, primarily ascorbic acid (6.0 to 8.2 mg g−1 fresh weight (FW)), flavonols (427.9 ± 0.4 μg g−1 FW) and antioxidant activity. We established a relationship between the antioxidant activity using Trolox equivalent antioxidant capacity (TEAC), cupric reducing antioxidant capacity (CUPRAC) and 2,2-diphenyl radical (DPPH) methods and the concentration of uncoloured compounds, such as flavonols and catechin. This antioxidant activity was primarily associated with the samples from Gorbea, Lonquimay, Loncoche, and Villarrica localities, and all of them were of the species Rosa rubiginosa L. The results here obtained represent novel information of rosehip fruits. In this sense, the reported information about compounds and antioxidant activities in rosehip fruits allowed us to continue new lines of research in relation to the potential formulation of new functional foods and also in the treatment and/or prevention of some diseases.
Einfluss von erhöhtem atmosphärischen CO2 auf die N2-fixierende Symbiose von Trifolium repens L. und Rhizobium leguminosarum biovar trifolii
(2007) Stöber, Sara; von Wirén, Nicolaus
CO2 is one of the main greenhouse gases strongly influencing the climate and the terrestrial ecosystem. Up to know little is known about the impact of elevated atmospheric CO2 on symbiotic interactions in the rhizosphere, especially on the N2-fixing symbiosis between Trifolium repens and Rhizobium leguminosarum biovar trifolii. First results of a ten-year Free-Air CO2 enrichment experiment (Swiss FACE) showed that after three years of CO2 fumigation the genetic composition of the Rhizobium population in the root nodules of T. repens had changed. The first part of this thesis set out to clarify the question whether a genetic difference in the Rhizobium population of root nodules of white clover could still be detected after ten years of CO2 fumigation or if an adaptation of the nodule bacteria to elevated CO2 concentrations had occurred. Furthermore the thesis addressed the question whether elevated atmospheric CO2 leads to quantitative and qualitative changes in the root exudation of T. repens particularly with regard to exudation of signal substances during the nodulation process. In summer 2002 white clover plants were collected from plots fumigated with CO2 and control plots of the Swiss FACE. Rhizobium strains were isolated from the clover root nodules and used for rep-PCR DNA fingerprinting. Results clearly showed that after ten years of CO2 enrichment changes in the genetic composition of the R. l. bv. trifolii could no longer be observed. Thus, CO2-induced changes in the population structure of rhizobia seemed to be transient. This can be traced back to the possibility that over the experimental period a new C/N equilibrium in the grassland ecosystem has been established. At the beginning of the FACE experiment an increase in the C/N ratio of the soil was detected, which could be balanced in the course of time through enhanced symbiotic N2 fixation and consequently a higher N input into the ecosystem. The observed stabilisation of the grassland ecosystem most likely caused a reduction of the indirect CO2 impact on the microorganisms. This might explain why a change in the genetic composition of Rhizobium strains was not longer detected after ten years in the Swiss FACE. To investigate an influence of elevated atmospheric CO2 concentration on the release of signalling compounds clover plants were cultivated hydroponically in two independent climate chamber trials under axenic and non-axenic conditions at ambient and elevated CO2 concentrations (400 and 800 ppm) and different levels of N supply. Root exudates were collected over a period of seven hours and at three and four different plant ages, respectively. Phenolic compounds were extracted by solid-phase extraction and afterwards analysed with HPLC and LC-MS. Additionally, the isolated fractions were tested for their ability to induce the nodulation genes of R. l. bv. trifolii using a nod-gene induction test. The CO2 enrichment caused an increase in shoot and root growth in both experimental setups, but did not provoke a change in the C/N ratio of the roots. Besides the known signal compound 7,4?-dihydroxyflavone new phenolic substances could be detected, which have not yet been described in literature. The fractions were identified by their polarity, light absorption and molecular weight as aglyca and flavones. All of these had the ability for nod-gene induction except one fraction (fraction 2). CO2 influenced the exudation of signalling compounds quantitatively but not qualitatively. The enhanced exudation, especially of 7,4?-dihydroxyflavone, could be attributed to the higher root mass under elevated CO2 but also to a higher release rate on a root fresh weight basis. The CO2 reaction of the clover plants, for the biomass production as well as for the root exudation, was clearly dependent on the N supply and only significant under axenic conditions. In individual cases the N impact was more pronounced than the CO2 effect: with increasing N demand axenic clover plants enhanced the exudation of the nod-gene inducing fraction C. It is concluded that this fraction, identified as a hydroxyflavone, has therefore an important signal function under N limitation. Besides the CO2 concentration and N supply, root exudation by T. repens was considerably influenced by the plant age, which caused a reduction of the signal exudation in older plants and qualitative changes of the released phenols, especially under non-axenic conditions. The present study suggests that the genetic shift of R. l. bv. trifolii detected at the beginning of the Swiss FACE experiment was most likely a consequence of the enhanced exudation of phenolic signal compounds of T. repens under elevated atmospheric CO2 concentrations.
Epidemiologische Aspekte der Falschen Mehltauinfektion durch Plasmopara viticola an Vitis
(2007) Keil, Sven; Spring, Otmar
The obligate biotrophic oomycete Plasmopara viticola (Berk. & Curt.) Berl. & de Toni causes downy mildew on grapevine. Plasmopara viticola is one of the economically most important pathogens in viticulture, with severe losses in yield of up to 70%. Existing prognosis models for plant protection in viticulture only allow yes/no statements on possible infection events in the vineyard. On the basis of these models, the severity of infections remain uncertain, although this represents a crucial point for an efficient application of fungicides. At low infestation severity, the application of protective fungicides at the end of the incubation period usually is sufficient. The low level of infestation can be tolerated and only further propagation of the pathogen must be prevented. In contrast, at high infestation severity curative fungicides have to be applied as soon as possible, because otherwise too much host tissue would be destroyed. Based on epidemiological studies and field experiments a prognostic concept has been designed, which enables the user to evaluate the relevance of infection events of grapevine downy mildew. This work has been carried out in the context of the research project ?Optimierung der Peronospora-Bekämpfung im Rebschutz auf der Basis eines erweiterten Prognosemodells (Forschungsvorhaben des Bundesministeriums für Ernährung, Landwirtschaft und Verbraucherschutz Nr. 514-33.54/01HS048)?. The developed concept was then integrated into the existing prognosis model and should support both consultants and winegrowers in using plant protection agents only in case of an expected increase of infestation frequency and severity. In this way, an effective and more economical use of fungicides is possible, which contributes to economic savings and reduces pesticide contamination of the environment. In the present study, aspects of sporangiogenesis, infestation of host tissue and hibernation, spreading of sporangia, interaction between vine leaves and sporangia as well as the climatic conditions during infection were analysed and evaluated with respect to the impact on epidemiology. The results improve existing literature data and deliver new insight to the epidemiology and biology of Plasmopara viticola.
Funktionelle Charakterisierung der Subtilase At4g21630 aus Arabidopsis thaliana
(2012) Knappenberger, Mathias; Schaller, Andreas
The goal of this thesis was the molecular and functional characterization of the A. thaliana subtilase At4g21630.
Heritable variation of foliar spectral reflectance enhances genomic prediction of hydrogen cyanide in a genetically structured population of eucalyptus
(2022) Ballesta, Paulina; Ahmar, Sunny; Lobos, Gustavo A.; Mieres-Castro, Daniel; Jiménez-Aspee, Felipe; Mora-Poblete, Freddy
Plants produce a wide diversity of specialized metabolites, which fulfill a wide range of biological functions, helping plants to interact with biotic and abiotic factors. In this study, an integrated approach based on high-throughput plant phenotyping, genome-wide haplotypes, and pedigree information was performed to examine the extent of heritable variation of foliar spectral reflectance and to predict the leaf hydrogen cyanide content in a genetically structured population of a cyanogenic eucalyptus (Eucalyptus cladocalyx F. Muell). In addition, the heritable variation (based on pedigree and genomic data) of more of 100 common spectral reflectance indices was examined. The first profile of heritable variation along the spectral reflectance curve indicated the highest estimate of genomic heritability (hg2=0.41) within the visible region of the spectrum, suggesting that several physiological and biological responses of trees to environmental stimuli (ex., light) are under moderate genetic control. The spectral reflectance index with the highest genomic-based heritability was leaf rust disease severity index 1 (hg2=0.58), followed by the anthocyanin reflectance index and the Browning reflectance index (hg2=0.54). Among the Bayesian prediction models based on spectral reflectance data, Bayes B had a better goodness of fit than the Bayes-C and Bayesian ridge regression models (in terms of the deviance information criterion). All models that included spectral reflectance data outperformed conventional genomic prediction models in their predictive ability and goodness-of-fit measures. Finally, we confirmed the proposed hypothesis that high-throughput phenotyping indirectly capture endophenotypic variants related to specialized metabolites (defense chemistry), and therefore, generally more accurate predictions can be made integrating phenomics and genomics.
Heterologe Expression und molekulare Charakterisierung des Dirigentproteins AtDIR6 aus A. thaliana
(2011) Pickel, Benjamin; Schaller, Andreas
Dirigent proteins are involved in the stereo- and regioselective control of plant secondary metabolism. Functionally described dirigent proteins from Forsythia intermedia and Thuja plicata couple coniferyl alcohol radicals to (+)-pinoresinol, a precursor of various lignans including the pharmaceutically relevant podophyllotoxin. The discovery of (?)-lariciresinol in A. thaliana roots and the accumulation of its precursor (?)-pinoresinol in a knock-out mutant lacking two pinoresinol reductases indicated the presence of a novel dirigent activity in A. thaliana which mediates the enantiocomplementary formation of (?)-pinoresinol. In this work AtDIR6 was identified as a candidate for this novel dirigent activity. The protein was cloned and heterologously expressed in a plant cell culture system. The recombinant protein was purified to appearent homogeneity by conventional chromatography methods. The purified protein was functionally active and directed the coupling of coniferyl alcohol radicals to (?)-pinoresinol in vitro. It was further shown that the stereoselectivity of AtDIR6 is opposed to that of known dirigent proteins in F. intermedia and T. plicata, and therefore, AtDIR6 is the first of the long-sought nantiocomplementary dirigent proteins. AtDIR6 was shown to possess a N-terminal signal peptide, which was cleaved during secretion between amino acids 29 and 30. Mature AtDIR6 accumulated extracellularly and remained non-covalently attached to the primary cell wall of suspension cultured cells. The native protein is glycosylated with two complex type and paucimannosidic Nglycans, respectively. It forms homodimers of app. 42 kDa and shows a high content of b-sheets. The functionally described dirigent proteins are small proteins that are characterised by the ability to bind coniferyl alcohol radicals and couple them enantiospecifically without possessing a catalytic activity of their own. Sequence identity between different dirigent proteins may be low. In these aspects dirigent proteins are similiar to lipocalins. A sequence alignment with dirigent proteins and lipocalins shows that the lipocalin-specific sequence motive, which is part of SCR II, is conserved among all functionally described dirigent proteins. Structural and mechanistic features of AtDIR6 suggest that dirigent proteins may belong to the calycin superfamily, which also includes lipocalins, and that their threedimensional structure may be that of a b-barrel.
Holzanatomische Veränderungen als Reaktion auf extreme Umweltereignisse in rezenten und subfossilen Eichen und deren Verifizierung im Experiment
(2014) Land, Alexander; Küppers, Manfred
This study focuses on changes in wood anatomy of recent (living study), container-grown (verification phase) and subfossil (subfossil study) oaks responding to severe biotic and abiotic events, as well as on “classical” tree-ring analysis (e.g. climate-growth relationships, signature-year analysis and age-trend analysis). Further, non-typical cell formations in wood anatomy with respect to the number, the shape and the size of xylem cells have been investigated. At this juncture, it was distinguished between continuous proxies (year-to-year), like total ring width or earlywood-vessel size, and non-continuous proxies (not year-to-year), like collapsed earlywood vessels or highly enhanced latewoodcell diameters. Measurement of annually resolved cross-section areas of earlywood vessels in recent and subfossil oak trees has been carried out by a specific developed semi-automatic tree ring image-analysis software (TRIAS). Basic objectives of this study were to connect the affecting biotic or abiotic events to specific changes in wood anatomy. Investigations on living (from locations with different side conditions in southern Germany) and container-grown (in the botanical garden, University of Hohenheim) oaks were performed to validate (verification phase) the results independently between the living and container study, getting clearer insights into the wood anatomical reaction of oaks and the homogeneity of the signal. Finally, the potential of qualitative and quantitative wood-anatomy for palaeo-climatology/-ecology is shown by applying the results to subfossil oaks (Holocene Oak Chronology Hohenheim). An event analysis was performed within two long-lasting periods (130 years and 1.000 years) in the mid-Holocene. Frequency distribution of extreme events, like high-magnitude floods or severe late-frost events, within these two periods in the mid-Holocene was compared with frequencies during the past 100 years. One result of this study is that one signature wood-anatomical change could be related to an extreme biotic or abiotic event, if the wood-anatomical feature occurred not only on local side level but on regional or national scale. Key evidence could be found for, that 1. out-of-true and collapsed earlywood vessels combined with traumatic tissue (wood-anatomical feature F/T) occurred after very low temperatures at the time of bud break, 2. intra-annual density bands are developed after rapid drops of temperature at an early stage (feature C) of the vegetation period or within (feature D) (depending on the position within the tree ring), 3. a long-lasting summer-flood event results in highly enhanced latewood-cell diameters (feature G), 4. a long-lasting spring-flood event reduces significantly the cross-section area of all vessels within earlywood (feature SEV), 5. latewood formation is absence or highly reduced in years with severe drought periods, 6. rapid drops of temperatures among winter periods (winter frost-events) reduce cross-section areas of initially built earlywood vessels (MVA5) and 7. continuous tree-ring proxies, like total ring width or earlywood-vessel variables, are significantly influenced by different climate factors. Wood-anatomical features from the recent study for 1., 3. and 4. could also be validated in the verification phase. The link between a biotic or abiotic event and the related specific wood-anatomical feature for 2., 5. and 6. could be found either in the recent study or the verification phase. Conducted measurements of diametrical stem growth, stem-growth dynamic and water deficit demonstrate big differences between the simulations (drought, frost, water logging, defoliation) that have been carried out on container-grown oaks. The simulations show massive changes in stem-growth dynamics of container-grown oaks affected by drought, frost and defoliation, whereas the waterlogged and control oaks reacted quite similar in their behaviour, and document the good adaptation of pedunculate oak trees to flooding. Pointer years were calculated and analysed for latewood and earlywood growth at the region Main and upper Rhine. Negative latewood-pointer years show great concurrence to years of annual summer-drought periods respectively long-lasting perennial droughts. Extreme events have been dated and analysed within two mid-Holocene periods of 2527-2396 BC (upper Rhine region) and 3279-2201 BC (Main region). High-magnitude floods and winter-frost events were dated using wood-anatomical features in subfossil oak trees (HOC Hohenheim). A “period of high river dynamic” in the Main valley could be analysed around 2750 BC. This result points out that the number of flood events in this period could be much higher than in the twentieth century whereas the number of detected winter-frost events at the upper Rhine region seems not to be increased compared to the past one hundred years.
Investigations into the secondary metabolite profile and bioactivity characteristics of various Achillea species grown under different environmental conditions
(2022) Salomon, Lysanne; Kammerer, Dietmar R.
Various Achillea species, including common yarrow Achillea millefolium L., are known to be rich in bioactive substances and are important medicinal plants in modern phytotherapy. In contrast, the alpine species Achillea atrata L. has hardly been studied so far regarding its bioactive compounds and the concomitant potential for human health. Having this in mind, the present work combined studies on the cultivation of A. atrata in different habitats with the phytochemical characterization of its secondary metabolite profile aiming at the generation of plant material with a defined and consistent phenolic profile suitable for pharmaceutical application. Particular emphasis was put on potential relationships between phytochemical profile and bioactivity, and these parameters were considered in comparison with other Achillea species. The results presented in this thesis reveal that the cultivation of A. atrata can be realized at sites differing significantly in altitude and, consequently, in their climatic conditions. Environmental conditions were found to have only a minor impact on the secondary metabolite profile. Rather, differences of various chemotypes might be more decisive. However, the contents of individual phenolic components, especially phenolic acids, is strongly dependent on environmental factors and reach their maximum at increasing altitude levels. Significant differences in the occurrence of the identified phenolic compounds were found between flowers and leaves. In addition to the previously mentioned studies, A. millefolium, Achillea moschata Wulfen, and A. atrata were compared with respect to their phenolic profile and antibacterial activity against gram-positive bacterial strains (Staphylococcus, Propionibacterium). A. atrata showed more similarities in the secondary metabolite profile with the alpine A. moschata than with A. millefolium. The two alpine Achillea species only differed in the occurrence of four compounds. All three aforementioned Achillea species reduced the growth of the tested bacteria. A. atrata showed the highest activity against Propionibacterium acnes and Staphylococcus epidermidis, which are involved in the development of the skin disease Acne vulgaris. In addition, A. atrata exhibited pronounced anti-MRSA potential. Bioassay-guided fractionation of the corresponding extracts revealed that the high antimicrobial activity was due to the flavonoids apigenin, centaureidin, and nevadensin, which were detected exclusively in the most polar fraction and were present in high amounts in A. atrata. Additional studies on the bioactivity and phytochemistry of A. atrata proved that this species exhibits higher antioxidant activity compared to A. millefolium, which was significantly correlated with its total phenolic content. Further bioassay-guided fractionation confirmed the correlation between the mentioned flavonoids in the most polar fraction and the high antioxidant capacity of A. atrata. In conclusion, A. atrata may be an alternative source for the development of novel herbal drugs, based on its compound and bioactivity characteristics.
Die linearen Drüsenhaare der Sonnenblume : Morphologie, Verbreitung, Metabolitprofil und Sesquiterpenbiosynthese
(2014) Aschenbrenner, Anna-Katharina; Spring, Otmar
The sunflower H. annuus and related species of Heliantinae possess, in addition to the very intensively studied capitate glandular trichomes, a second type of multicellular and biosynthetically active trichome which was so far underinvestigated. In the present work, these linear glandular trichomes (LGT) were studied in detail. Microscopic investigations revealed a wide distribution of LGT within sunflower and species of the subtribe Heliantinae. LGT occurred on leaves and stems of sunflower, but the highest accumulation was found at the involucral bracts of the flower head. This suggests that they may play a chemoecological role in plant-insect or plant-herbivore interaction. The fast development of LGT within only 32h was found to take place during the emergence of leaf primordia on seedlings between the second and third day of germination. The biosynthetic activity got visible with the accumulation of substances successively in the tip cell and progressed towards the trichome base. The deposition of metabolites was accompanied by a loss of cellular vitality and was indicated by the degradation of the nucleus. Through examining the metabolite profile of LGT, it was revealed that they contain oxidized flavones. These compounds were subsequently isolated and structurally characterized. In addition, a wide variety of bisabolene-type sesquiterpenes was found. Among the identified structures were several Glandulones and Heliannuoles already known from previous metabolic studies of sunflower, but also several new compounds. Since the biosynthesis of these compounds from sunflower is fairly unknown, the focus of the present study was on the discovery and characterization of key enzymes that catalyze the first step of bisabolene biosynthesis. From the transcriptome of LGT, candidate genes for the formation of bisabolenes were identified by homology-based experiments. The complete sequences of the transcribed genes from cDNA was isolated by RACE experiments and confirmed by comparison with genomic sequences from the sunflower genome project. The functional identity of two candidate genes was achieved by means of heterologous expression in a yeast system specialized for the characterization of sesquiterpenes. The product of the transformed yeast cells was isolated and spectroscopically identified as cis-γ-bisabolene. Based on these experiments, these two enzymes could be identified as isoforms of a cis-gamma-bisabolene synthase, and represents the first known enzymes of this type characterized in sunflower.
Lipid remodeling of contrasting maize (Zea mays L.) hybrids under repeated drought
(2023) Kränzlein, Markus; Schmöckel, Sandra M.; Geilfus, Christoph-Martin; Schulze, Waltraud X.; Altenbuchinger, Michael; Hrenn, Holger; Roessner, Ute; Zörb, Christian
The role of recovery after drought has been proposed to play a more prominent role during the whole drought-adaption process than previously thought. Two maize hybrids with comparable growth but contrasting physiological responses were investigated using physiological, metabolic, and lipidomic tools to understand the plants’ strategies of lipid remodeling in response to repeated drought stimuli. Profound differences in adaptation between hybrids were discovered during the recovery phase, which likely gave rise to different degrees of lipid adaptability to the subsequent drought event. These differences in adaptability are visible in galactolipid metabolism and fatty acid saturation patterns during recovery and may lead to a membrane dysregulation in the sensitive maize hybrid. Moreover, the more drought-tolerant hybrid displays more changes of metabolite and lipid abundance with a higher number of differences within individual lipids, despite a lower physiological response, while the responses in the sensitive hybrid are higher in magnitude but lower in significance on the level of individual lipids and metabolites. This study suggests that lipid remodeling during recovery plays a key role in the drought response of plants.
Mobile signals in plant parasitism
(2024) Greifenhagen, Anne; Schaller, Andreas
Close to two percent of all flowering plants evolved parasitism, with some parasitic species, like Striga spp. from the Orobanchaceae family, posing a prevailing threat to crop yield. Parasitic weed management is challenging and requires a deeper understanding of the complex parasite-host relationship (Section 1.1). Parasitic plants infect and parasitize host plants through a multicellular feeding organ, the haustorium. This organ may either develop from the root tip as a single terminal haustorium or emerge multiple times along the growing roots, called lateral haustoria. In both cases, protohaustoria develop into mature haustoria that enable the withdrawal of water and nutrients. Parasitism depends on parasite and host endogenous signaling but also on communication between both partners (Section 1.2). This is similar to the development of other plant organs like lateral roots and symbiotic nodules, whose number is adjusted by an autoregulation of nodulation (AON) system. The induction of parasitic organs by pathogenic nematodes, but in particular also by parasitic plants, involves the manipulation, neofunctionalization, and interspecific exchange of mobile signals (Section 1.3). However, most of these molecular cues remain elusive in the parasitic plant-host plant interaction. This work aimed to address the biogenesis and function of parasite-derived endogenous and interspecific mobile signals involved in early till late stages of parasitism in the model system Phtheirospermum japonicum infecting Arabidopsis thaliana (Section 1.4). Transcriptome and genome studies on parasitic plants paved the way to unravel signaling cues contributing to parasitism. The evolution of parasitism correlates with the expansion of certain gene families followed by their parasitism-related neofunctionalization as seen for the KARRIKIN-INSENSITIVE 2 ‘divergent’-type (KAI2d) gene family in parasitic plants of the Orobanchaceae. Likewise, subtilisin-like serine protease (subtilase, SBT) genes in P. japonicum and Striga underwent an expansion, and some show haustorium tip-specific expression. The proteolytic activity of PjSBTs is required for proper haustorium formation and development. Despite their importance, no substrates of PjSBTs have been identified. In this work, PjSBT1.2.3 was found to be co-expressed with CLAVATA3(CLV)/EMBRYO-SURROUNDING REGION-related 3 (PjCLE3) during infection in the same domain of the haustorium. PjSBT1.2.3 cleaved PjCLE3 in vitro, thereby releasing the bioactive mature PjCLE3 peptide (Section 2.1, Fig.2.1.1). Sensing host-derived haustorium-inducing factors (HIFs) initiates haustorium organogenesis. In the absence of a host, the synthetic mature PjCLE3 induced protohaustorium formation similar to a host-derived benzoquinone HIF. Combined treatment of both HIFs potentiated their activity (Section 2.1, Fig.2.1.2). Pj cle3 knock-out hairy roots (HRs) formed fewer haustoria, particularly due to the absence of secondary protohaustoria (Section 2.1, Fig.2.1.3). These data demonstrate the existence of an autoregulation of haustoria formation (AOH) system as part of which the PjSBT1.2.3-PjCLE3 module, in analogy to AON, regulates the number of P. japonicum lateral haustoria. During the early stages of parasitism, the parasitism-related PjSBT1.2.3-PjCLE3 module promotes protohaustorium formation by sensitizing the parasite root for host-derived HIFs (Section 3.1). A homologous SBT-CLE module also exists in Striga, even though the parasite forms a terminal haustorium. Striga CLE2s are identical to host CLEs and this mimicry might improve nutrient allocations from the host during later stages of parasitism (Section 2.1, Fig.S2.1.2; Section 3.2). Similarly, parasite-derived cytokinin (CK) translocates through the haustorium inducing host hypertrophy, a swelling of host tissue above the infection site, thereby potentially benefiting parasite nutrient acquisition. In agrobacteria and plants, isopentenyltransferases (IPTs) synthesize CK precursors. Similar to SBTs and KAI2ds from parasitic plants, P. japonicum and S. hermonthica IPT1 genes exist as multiple copies, with one copy, PjIPT1a showing specific expression at the tip of haustoria (Section 2.2, Fig.2.2.1, Fig.2.2.2). Bioinformatic tools predicted a chloroplast transit peptide (CTP) for PjIPT1s, but PjIPT1-GFP fusions localized to cytoplasm and nucleus suggesting the CTP to be non-functional (Section 2.2, Fig.2.2.2, Fig.S2.2.2). To test substrate specificity and activity of both PjIPT1 copies, isoprenylation-activity was probed in vitro. PjIPT1b used both AMP and ATP as substrates, whereas PjIPT1a displayed a higher affinity for AMP, indicating that PjIPT1b may be the canonical, whereas PjIPT1a is the parasitism-related IPT (Section 2.2, Fig.2.2.4). This is further supported by the observation that CRISPR/Cas9-mediated mutation of PjIPT1a abolishes CK responses in the infected host (Section 3.3, Section 2.2, Fig.2.2.3). SBT-CLE, IPT-CK together with KAI2ds all have in common that their parasitism-related function may evolutionally result from gene duplication combined with neofunctionalization. Targeting duplicated and neofunctionalized genes may prove to be a promising strategy to combat parasitic weeds. (Sections 3.4, 3.5).
Molekulare und entwicklungsbiologische Charakterisierung von Schlüsselenzymen der Naturstoffbiosynthese in Drüsenhaaren der Sonnenblume
(2008) Göpfert, Jens C.; Spring, Otmar
Glandular trichomes from anther appendages of sunflower were collected and their RNA was isolated. Sequence comparison with known plant sesquiterpene synthases was used to identify sunflower synthases in RT-PCR reactions. Three enzymes, HaGAS1, HaGAS2 and HaCS with high similarities to already characterized sesquiterpene synthases were identified. Their nucleotide sequences were completely established on the genomic level and as RNA transcripts. The nucleotide sequences as well as the deduced amino acid sequences showed typical characteristics of terpene synthases. In order to characterize the enzymes, the sesquiterpene synthase genes were cloned and expressed in E. coli. In vitro assays with the recombinant enzymes were carried out using the native substrate farnesyldiphosphate. The resulting products were extracted and analysed by GC-MS. They were identified by comparison of data base MS-data and using reference samples under identical analytical conditions. Two expressed enzymes, HaGAS1 and HaGAS2, synthesized germacrene A as a single product. Heterologous in vivo expression of both germacrene A-synthases in S. cerevisiae confirmed the in vitro result, since the analysis of the synthesized product showed a single germacrene A peak. Due to a very low in vitro activity of HaCS, the products of the third synthase could not be directly determined by MS-analysis. Therefore, the enzyme was expressed as a thioredoxin-fusion protein in vivo in transgenic yeast. This attempt resulted in a much higher rate of product yield. Two main and at least six minor products were traced in GC-analysis. They were confirmed as sesquiterpene hydrocarbons by GC-MS analysis. One of the two main products was identified as gamma-cadinene, whereas the second main peak could not be determined conclusively. Among the minor compounds alpha-copaene, alpha-muurolene und beta-caryophyllene were identified. Screening of a H. annuus EST library (established at the Berkeley Center for Synthetic Biology, University of California, Berkeley, USA) from mRNA of trichomes revealed the presence of a cytochrome P450 protein which showed high similarity to an Artemisia annua enzyme involved in artemisinic acid biosynthesis. This enzyme and another similar protein from Lactuca sativa were cloned and coexpressed with the germacrene A-synthase HaGAS2 in yeast. The resulting product was indirectly determined as germacrene A carboxylic acid using GC-MS analysis. These novel cytochrome P450 enzymes from sunflower and lettuce can be characterized as multifunctional germacrene A-monooxygenases. They catalyse a three-step oxidation leading from germacrene A to germacrene A carboxylic acid. This oxidation process represents an essential step towards the biosynthesis of sesquiterpene lactones. Semiquantitative RT-PCR analysis demonstrated that the expression of all three sesquiterpene synthases and the sunflower P450 monooxygenase occurred directly within trichome cells. The expression was highly upregulated during the secretory stage of the capitate glandular trichomes. This developmentally regulated expression was shown for the first time in trichomes. Additionally to sesquiterpene synthase activity in trichomes of anthers and leaves, it also was detected in sunflower roots. In addition, 5-deoxynevadensin was identified as a new constituent of the glandular trichomes of sunflower. This 5-deoxy-flavone is responsible for the bright blue fluorescence of sunflower trichomes detected by fluorescence microscopy. The newly identified component may act as protectant for the STL against UV-degradation.
Neue Cytochrom P450 Enzyme des Sesquiterpenlacton Stoffwechsels der Sonnenblume (Helianthus annuus L.)
(2016) Frey, Maximilian; Spring, Otmar
In the present work additional steps towards the elucidation of the biosynthetic pathway of H. annuus sesquiterpene lactones (STL) were achieved. Firstly candidate sequences were retrieved from a transcriptome database by filtering according to expression pattern and similarity to P450 enzymes known to participate in STL biosynthetic pathways. Open reading frames (ORFs) were obtained using 3´-and 5´-RACE-PCR. Previously described and newly identified candidate genes were then transformed in yeast vectors and expressed in combination with different substrate vectors. A high throughput micro approach was developed that allowed the expression and analysis of many yeast strains at the same time. For the transient expression in N. benthamiana the genes of known and putative enzymes were introduced via Agrobacterium mediated transformation. Using the in planta expression system the complete STL pathway of sunflower to costunolide was reconstructed de novo in a step-by-step approach. Previously described Michael-addition reactions of α-methylene-γ-lactone type STL to the thiol group of cysteine or glutathione in tobacco expression systems could be observed for all STL investigated. Chemically synthesized STL adducts were used as reference for the identification of in planta produced STL adducts. Enzyme characterization was conducted in two different in vivo expression systems, in yeast (Saccharomyces cervisiae) and tobacco (Nicotiana benthamiana). For the investigated biosynthetic pathway, differences between these two expression systems were discussed. Candidate gene M4 showed an unexpected product in yeast (farnesyl-δ-lactone) and led in combination with HaG8H to the production of costunolide. In the plant expression system, germacrene A acid was converted to costunolide by M4 in the absence of HaG8H. In both cases, M4 was involved in the synthesis of costunolide and should therefore be assigned Helianthus annuus costunolide synthase the underlying reaction mechanism should however be investigated more thoroughly. Helianthus annuus costunolide 14-hydroxylase HaC14H (candidate M33) was characterized in yeast and tobacco. A classification into subfamily CYP71CB, together with Tp8878 the Tanacetum parthenium costunolide/parthenolide 3β-hydroxylase is proposed. It was shown that in planta the main product of HaG8H exists most likely as inunolide, which would be the entry point for the biosynthesis of 8-epixanthatine and tomentosine. Candidate S2 from Ikezawa et al. (2011) was found to convert 8β-hydroxy-germacrene A acid to 8β-hydroxy-costunolide (eupatolide) in tobacco, but not in yeast, producing several byproducts. The name Helianthus annuus eupatolide synthase HaES is proposed accordingly. HaES has 47 % amino acid identity to the parthenolide synthase from T. parthenium (TpPTS). A classification into a new CYP71 subfamily is proposed. Two alternative metabolic routes led to 8β-hydroxy-costunolide in the expression studies in tobacco, the underlying mechanisms are discussed. Enzymes involved in STL biosynthesis were expressed in inner tissues of young Helianthus annuus plants; the induction of expression of STL biosynthesis enzymes in leaf primordia correlates with the development of capitate glandular trichomes (CGT) and STL synthesis. HaC14H was found in a chromosomal region in proximity to several P450 enzyme candidates, that share the same subfamily and the expression in capitate glandular trichomes (CGT). Therefore involvement of these enzymes in later steps of the biosynthesis of the elaborate STL structures found in CGT is likely.
PEP7 is a ligand for receptor kinase SIRK1 to regulate aquaporins and root growth
(2021) Wang, Jiahui; Schulze, Waltraud
Receptor kinases constitute the largest protein family in regulating various responses to external and internal biotic and abiotic signals. Functional characterization of this large protein family and particularly the identification of their ligands remains a major challenge in plant biology. Previously, we identified SIRK1 and QSK1 as a receptor / co-receptor pair involved in regulation of aquaporins in response to osmotic changes induced by sucrose. Here, we now identify a member of the Elicitor Peptide (PEP) family, namely PEP7, as a ligand to receptor kinase SIRK1. PEP7 was shown to bind to the extracellular domain of SIRK1 with a binding constant of 19 µM. PEP7 was secreted to the apoplasm specifically in response to sucrose. Formation of a signaling complex involving SIRK1, QSK1 as well as aquaporins as substrates was induced by sucrose or external PEP7 treatment. PEP7 induced aquaporin phosphorylation and water influx activity. The knock-out mutant of receptor SIRK1 was not responsive to external PEP7 treatment. Binding to receptor SIRK1 and induction of physiological responses was specific to PEP7, neither other members of the PEP-family (PEP6, PEP4), nor other small signaling peptides (CLEs, IDA, RALFs) induced SIRK1 kinase activity, aquaporin phosphorylation, or protoplast water influx activity.
Peru Botanische Exkursion 2008
(2010) Dinter, Ina; Zimmermann, Reiner
The botanical excursion to Peru, a global hotspot of biodiversity, provides an overview of the climatic, regional and plant ecological situation of the equatorial neotropics. The areas visited cover the hyper arid coastal deserts, the dry and cold andine Puna, the Amazon lowland rainforests and the seasonal dry forests of North Peru. The excursion report presented was compiled from presentations and protocols of the participants and contains the list of plants found at each site visited.
Phänotypische und molekulare Analyse von Kreuzungsnachkommen auf Resistenz gegen Ährenfusariosen bei Triticale (x Triticosecale Wittmack)
(2011) Großmann, Maren; Großmann, Maren
Fusarium head blight (FHB) is an epidemic disease of cereals but disease control still is insufficiently possible. Resistance breeding can be addressed as one approach to reduce the mycotoxin contents. Since 2006 the European Union had strict rules governing the values for Desoxinivalenol (DON) in food products. The extraordinary loads through mycotoxins in triticale, which are caused by FHB, lead to large reductions in grain yield and quality. Beyond the fact that it can cause health issues in animals, especially in pig feed, it may induce financial burdens for farming industries. Resistance against FHB is inherited quantitatively with mainly additive effects and therefore is only recorded by complex field experiments. Molecular markers accelerate resistance breeding and enable specific introgressions of favorable QTL. The main topic of this thesis is to show the application possibilities of molecular markers for the investigation of FHB resistance in triticale. In detail this study aims to analyze factors leading to a minimized mycotoxin accumulation in perennial field trials at several locations. Furthermore genetic maps of two different crossing populations were compiled and QTL mapping for ear appearance, plant height, DON content and FHB resistance was implemented. For all populations significant varieties had been displayed for all characteristics. The average values for FHB rate were between 8 and 43 % depending on year and location. The heritability?s showed high values for each population (h2 = 0.7 ? 0.82). The substantial genotype-environment-interaction pointed out the importance of field experiments. Furthermore no significant correlation was obtained between ear appearance and plant height. Due to the fact that the correlation factor between DON-content and FHB rate was very low (r = 0.32) to moderate (r = 0,65). Therefore no assumption about the DON-content subjected to the FHB rate could be made. The mapping of both populations LASKO x ALAMO and LASKO x TRIMESTER has been accomplished with SSR- and DArT-markers. The rate of polymorphism could be increased from 7 - 12 % respectively to 9 - 18 %. Genetic maps were constructed with length of 1.815 and 1.407 cM. They indicated 5.19 and 4.54 cM in their average marker distances. Throughout the QTL mapping several QTL were detected (6 QTL for ear appearance, 4 QTL for plant height, 1 QTL for DON-content and 4 QTL for FHB rate). These QTL explained 8 to 66 % of the phenotypical variance. In addition crossing populations of LASKO x TRIMESTER showed 2 QTL for ear appearance, 1 for plant height and 2 QTL for FHB resistance, which could explain 3 to 41 % of the phenotypical variance. This was also observed in several other QTL-surveys for wheat. Prospectively using molecular markers and genetically engineered methods will increase the research and development of resistant varieties and lead to a reduced mycotoxin accumulation. So far neither genetic maps nor any QTL studies for FHB resistance or DON-content in triticale have been published. Due to the fact that resistance is transmitted by several genes numerous artificial inoculations have to be carried out during breeding. Additive gene effects are useful to combine several resistance genes from different parents. Established methods in plant breeding such as recurrent selection, phenotypical and marker-based selection are successfully in use to reduce FHB- symptoms and to reduce the mycotoxin value but these methods have to be improved. The results of this thesis are promising for a superior resistance breeding in the future.
Plasmopara viticola, the downy mildew of grapevine : phenotypic and molecular characterization of single sporangium strains infecting hosts with different resistance levels
(2015) Gómez Zeledón, José Javier; Spring, Otmar
The downy mildew of grapevine, Plasmopara viticola, is one of the most important pathogens in viticulture. Its genetic diversity had been assessed in some previous studies using molecular markers, but the diversity of the infection behavior has not yet been addressed adequately. Therefore, the development of a fast, reliable and uncomplicated assay to screen for pathogen phenotypes on host with different resistance levels was a major task of this work. A leaf disc test was proposed, evaluating sporulation and necrosis produced by the pathogen on Vitis plants with different susceptibility. Using this bioassay, interesting strains were assessed and kept for future studies. The urgent need to work with genetic homogeneous inoculum was shown, because the assays revealed a high phenotypic diversity in isolates collected from the field as a bulk sample. Hence, a cloning technique to obtain single sporangium strains was found useful to avoid working with mixed genotypes. The leaf disc bioassay also allowed screening for fungicide resistance in P. viticola populations. Isolates resistant to dimethomorph and metalaxyl, two important fungicides for oomycetes control, were detected. Higher resistance was associated with fields were the fungicide application was high as well. Some strains were even resistant to doses where the fungicide exhibits phytotoxic activity to grapevine. The approach of characterizing P. viticola pathotypes on different host plants of Vitis vinifera cultivars and Vitis species from North America and Asia revealed a broad spectrum of fully susceptible to completely resistant reactions. This information is of direct practical value in future plant breeding programs, but also provides the chance to select specific host-pathogen combinations to study the mechanisms of resistance or susceptibility. Fluorescence microscopy revealed how the infection progress of highly and lowly virulent strains advance in tolerant and susceptible hosts, and which points of the infection are interesting for future studies. On the molecular level, effectors were investigated to trace their possible involvement in the infection process. It was found that RXLR 1, NLP 1, Elicitin like 2, Glucanase inhibitor 2 and 4 , and 1,3-ß Glucanase 2 are candidates which are upregulated in the earliest infection stages. Following the here established methodology and suggested strategy it should be possible in the future to get a better insight in the mechanisms of infection and resistance of grapevine downy mildew.
Plastid phylogenomics reveals evolutionary relationships in the mycoheterotrophic orchid genus Dipodium and provides insights into plastid gene degeneration
(2024) Goedderz, Stephanie; Clements, Mark A.; Bent, Stephen J.; Nicholls, James A.; Patel, Vidushi S.; Crayn, Darren M.; Schlüter, Philipp M.; Nargar, Katharina; Goedderz, Stephanie; Australian Tropical Herbarium, James Cook University, Cairns, QLD, Australia; Clements, Mark A.; Centre for Australian National Biodiversity Research (joint venture between Parks Australia and CSIRO), Canberra, ACT, Australia; Bent, Stephen J.; Data61, Commonwealth Industrial and Scientific Research Organisation (CSIRO), Brisbane, QLD, Australia; Nicholls, James A.; Australian National Insect Collection, Commonwealth Industrial and Scientific Research Organisation (CSIRO), Canberra, ACT, Australia; Patel, Vidushi S.; National Research Collections Australia, Commonwealth Industrial and Scientific Research Organisation (CSIRO), Canberra, ACT, Australia; Crayn, Darren M.; Australian Tropical Herbarium, James Cook University, Cairns, QLD, Australia; Schlüter, Philipp M.; Department of Plant Evolutionary Biology, Institute of Biology, University of Hohenheim, Stuttgart, Germany; Nargar, Katharina; Australian Tropical Herbarium, James Cook University, Cairns, QLD, Australia
The orchid genus Dipodium R.Br. (Epidendroideae) comprises leafy autotrophic and leafless mycoheterotrophic species, with the latter confined to sect. Dipodium . This study examined plastome degeneration in Dipodium in a phylogenomic and temporal context. Whole plastomes were reconstructed and annotated for 24 Dipodium samples representing 14 species and two putatively new species, encompassing over 80% of species diversity in sect. Dipodium . Phylogenomic analysis based on 68 plastid loci including a broad outgroup sampling across Orchidaceae found that sect. Leopardanthus is the sister lineage to sect. Dipodium. Dipodium ensifolium , the only leafy autotrophic species in sect. Dipodium , was found to be a sister to all leafless, mycoheterotrophic species, supporting a single evolutionary origin of mycoheterotrophy in the genus. Divergence-time estimations found that Dipodium arose ca. 33.3 Ma near the lower boundary of the Oligocene and that crown diversification commenced in the late Miocene, ca. 11.3 Ma. Mycoheterotrophy in the genus was estimated to have evolved in the late Miocene, ca. 7.3 Ma, in sect. Dipodium . The comparative assessment of plastome structure and gene degradation in Dipodium revealed that plastid ndh genes were pseudogenised or physically lost in all Dipodium species, including in leafy autotrophic species of both Dipodium sections. Levels of plastid ndh gene degradation were found to vary among species as well as within species, providing evidence of relaxed selection for retention of the NADH dehydrogenase complex within the genus. Dipodium exhibits an early stage of plastid genome degradation, as all species were found to have retained a full set of functional photosynthesis-related genes and housekeeping genes. This study provides important insights into plastid genome degradation along the transition from autotrophy to mycoheterotrophy in a phylogenomic and temporal context.
Radial tree growth dynamics and xylem anatomy along an elevational gradient in the El Sira Mountains, Peru
(2019) Niessner, Armin; Küppers, Manfred
The explicit purpose of this study was to (1) characterize climate and vegetation along the western slope of the Cerros del Sira (Peru), (2) evaluate radial tree growth along this slope in response to seasonal rainfall anomalies, (3) reveal how the meteorological environment controls tree radial stem growth, and (4) to investigate how xylem anatomy relates to dynamics of tree growth. From May 2011 until September 2015, radial stem growth of 67 trees was monitored using point-dendrometers, and meteorological parameters were measured within five sites along an elevational gradient. Additionally, tree dimensions (stem diameter, stem height) and xylem anatomical traits (mean vessel diameter, vessel frequency, cumulative vessel area and potential hydraulic conductivity) were determined. The transect extends from lowland terra firme forests (ca. 250 m asl) over submontane forests, late and mid successional montane cloud forests up to exposed elfin forests (ca. 2200 m asl). Continuous rainfall records for remote tropical areas are extremely rare and measurements along this transect are also incomplete. Monthly rainfall estimates by the TRMM PR satellite ("product 3B42") were highly correlated with rain gauge observations, although they underestimate rainfall at high elevations. Different intra-annual tree growth patterns could be identified within each elevational forest type, showing species/individuals with seasonally independent continuous or alternating growth patterns and strictly seasonal growth. Stem growth at each site was typically higher during rainy seasons, except for in the elfin forest. The rainy season from October 2013 to March 2014 was extraordinarily dry, with only 73 % of long-term mean precipitation received, which resulted in reduced tree radial growth rates, again with the exception of the elfin forest. Different analytical approaches revealed that precipitation is the main growth-controlling factor at lower elevations, especially during rainy seasons. Growth within montane and cloud forests positively correlates to solar radiation. Tree growth within the elfin forest is only weakly correlated to meteorological parameters, but dry conditions during dry seasons promote growth. It was hypothesized that (1) individuals/species with large vessel diameters and low vessel frequencies have higher radial growth rates, but are more vulnerable to relatively dry periods. Therefore, (2) they are more likely to grow seasonally and predominantly during rainy seasons, (3) their growth during the exceptionally dry rainy season 2013/14 was more constrained, and (4) their growth is generally closer correlated to meteorological parameters. Larger trees tend to have larger vessel diameters, which positively relates to radial growth rates, and they also tend to grow more seasonal. As hypothesized, trees having large vessel diameters are more sensitive to droughts, as indicated by stronger positive correlations with rainfall and negative with solar radiation. However, in mountainous forests, relations between xylem anatomical traits and tree growth dynamics seem to be more complex. In late-successional cloud forests, growth of trees with large vessel diameter is positively, while of trees with small vessel diameter negatively related to solar radiation. Sensitivity to the dry rainy season 2013/14, expressed as relative reduction in growth compared to "normal" rainy seasons, could not be explained by xylem anatomical traits, contradicting the preceded hypothesis. Tropical lowland rainforest species, especially individuals with large vessels, will likely suffer from increasing drought frequencies and intensities. How montane forest ecosystems will react to a (globally) changing climate is rather uncertain, especially in exposed elfin forests. Results of this study suggest that species of late-successional tropical montane forests may profit from higher temperatures. While montane tropical rain forests may also suffer from prolonged droughts, exposed ridges covered by elfin forests still receive plenty of precipitation and may benefit from receiving more solar radiation for photosynthesis and, thus, grow faster.
Strategies and mechanisms of cellular interaction between the parasitic weed Orobanche cumana WALLR. and its host Helianthus annuus L.
(2020) Krupp, Anna Clarissa; Spring, Otmar
Sunflower broomrape, Orobanche cumana WALLR., is a root parasitic plant causing considerable yield losses in sunflower cultivation in Europe, North Africa and Asia. Comprehensive knowledge about early interaction stages between host and parasite is necessary to find new ways of controlling this weed. In this thesis, three aspects regarding the biology of O. cumana were studied: 1) the chemotropism of O. cumana germtubes which bend towards the host root, 2) the development of O. cumana on resistant and susceptible sunflower lines and 3) the development of the phloem connection between the O. cumana haustorium and the sunflower host root. Sesquiterpene lactones in sunflower root exudates act as germination stimulants for O. cumana. As sesquiterpene lactones are known inhibitors of plant elongation growth and seem to play a role in the phototropic curvature of sunflower hypocotyls, a chemotropism bioassay on water agar was established to test if they also serve as chemotropic signals for the host-finding of O. cumana germtubes. When sesquiterpene lactone containing sunflower root exudate, sunflower seed oil extract or the sesquiterpene lactone reference costunolide were applied on filter discs, 70 % of the germtubes showed orientation towards them. The artificial strigolactone GR24, however, did not induce chemotropism. A concentration gradient of sesquiterpene lactones exudated from the host root is likely to be responsible for a stronger inhibition of elongation growth on the host-facing flank of the germtube. This would confer a double role of sesquiterpene lactones from root exudates in the sunflower-broomrape-interaction, namely as germination stimulants and as chemotropic signals. One way of controlling O. cumana is the cultivation of resistant sunflower lines. However, this resistance is rapidly overcome by more aggressive pathotypes of the parasite. Therefore, the resistance or tolerance reaction of the sunflower genotype T35001 was investigated in comparison to six other sunflower genotypes with different resistance characteristics. The development of O. cumana was monitored in a root chamber system which allowed permanent assessment of germination, attachment and tubercle formation in the different host-parasite-combinations. All seven tested sunflower lines induced germination and attachment of O. cumana, independent of the expected resistance or susceptibility of the host. A difference between compatibility or incompatibility of the interactions was only observed at the tubercle stage. On T35001, tubercles never occurred, neither in root chamber nor in pot experiments. To find out why the development stopped before the tubercle stage, samples of sunflower roots with attached O. cumana seedlings were analysed by bright field-, fluorescence- and transmission electron microscopy. Histological studies revealed that O. cumana penetrated the host root, but never reached the host’s vascular bundle. The root cortex cells surrounding the Orobanche haustorium showed no ultrastructural changes such as cell wall thickening. Fluorescence microscopy revealed no callose depositions or signs of phytoalexin release. However, ultrastructural examination of the host-parasite-interface showed degeneration processes in both cortex and haustorial cells. Cortex cells were flooded with bacteria, haustorium cells showed degeneration of cytoplasm and nuclei. The resistance mechanism that prevented further development of the O. cumana haustorium did not express itself in a histologically visible way. As holoparasite, O. cumana acquires its entire demand for water, minerals and organic nutrients from the host’s vascular system. The development of the xylem connection between O. cumana and sunflower had previously been reported, but the phloem connection is far more relevant for the parasite in terms of organic nutrients. Accordingly, the ultrastructure of the phloem connection between the haustorium of young O. cumana tubercles and the sunflower root was examined. Parasite and host tissues were intermingled at the contact site and difficult to distinguish, but sieve-tube elements of O. cumana and sunflower could be differentiated according to their plastid ultrastructure. While sieve-element plastids of O. cumana were larger, often irregular in shape and contained few, small starch inclusions, sieve-element plastids of the host were significantly smaller, always round with more and larger starch inclusions. This made it possible to trace the exact contact site of host and parasite sieve elements to show a direct symplastic phloem connection between the two species. The interspecific sieve plate showed more callose on the host side. This allowed detection of newly formed plasmodesmata between host sieve-tube elements and parenchymatic parasite cells, thus showing that undifferentiated cells of the parasite can connect to fully differentiated sieve elements of sunflower.

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