Browsing by Person "Ludewig, Uwe"

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Ammonium fertilization increases the susceptibility to fungal leaf and root pathogens in winter wheat
(2022) Maywald, Niels Julian; Mang, Melissa; Pahls, Nathalie; Neumann, Günter; Ludewig, Uwe; Francioli, Davide
Nitrogen (N) fertilization is indispensable for high yields in agriculture due to its central role in plant growth and fitness. Different N forms affect plant defense against foliar pathogens and may alter soil–plant-microbe interactions. To date, however, the complex relationships between N forms and host defense are poorly understood. For this purpose, nitrate, ammonium, and cyanamide were compared in greenhouse pot trials with the aim to suppress two important fungal wheat pathogens Blumeria graminis f. sp. tritici (Bgt) and Gaeumannomyces graminis f. sp. tritici (Ggt). Wheat inoculated with the foliar pathogen Bgt was comparatively up to 80% less infested when fertilized with nitrate or cyanamide than with ammonium. Likewise, soil inoculation with the fungal pathogen Ggt revealed a 38% higher percentage of take-all infected roots in ammonium-fertilized plants. The bacterial rhizosphere microbiome was little affected by the N form, whereas the fungal community composition and structure were shaped by the different N fertilization, as revealed from metabarcoding data. Importantly, we observed a higher abundance of fungal pathogenic taxa in the ammonium-fertilized treatment compared to the other N treatments. Taken together, our findings demonstrated the critical role of fertilized N forms for host–pathogen interactions and wheat rhizosphere microbiome assemblage, which are relevant for plant fitness and performance.
Bildung und Forschung im Kontext der digitalen und ökologischen Transformation des Agrarbereichs im Banat und Baden-Württemberg - auf dem Weg zu Ressourceneffizienz und Resilienz
(2023) Weinmann, Markus; Landtag und Staatsministerium Baden-Württemberg , Universität für Lebenswissenschaften " König Michael I " in Timisoara; Raupp, Manfred G.; Ludewig, Uwe; Flad, Angelika
The Banat Green Deal project "GreenERDE" (Education and Research in the context of the digital and ecological transformation of agriculture in the Banat Region and Baden-Württemberg - towards resource efficiency and resilience) aimed to strengthen the the competitiveness of the agricultural sector in the Romanian Banat, Baden-Württemberg and neighboring regions with innovative technical and at the same time socio-cultural connect interesting content. The advanced training program “Farming in Responsibility for Our Common World” carried out as part of this project aims at the transfer of knowledge and experience among farmers and other interested persons. Current and future challenges, such as ecological conversion and the digital transformation of agricultural production, but also social, economic and cultural aspects were addressed. Innovative and relevant knowledge from practice, research or development projects throughout Europe and other continents is presented.
Comprehensive analyses of DNA methylation profile, regulation on flowering, and seed mineral accumulation in Arabidopsis thaliana in response to zinc deficiency
(2016) Chen, Xiaochao; Ludewig, Uwe
Zinc (Zn) is an essential micronutrient for plant growth and development, which plays important roles in DNA binding, metabolic, catalytic and transcriptional regulator activities. However, Zn deficiency is a worldwide problem due to its limited bioavailability in soils in many agricultural areas, often as a result of high CaCO3 content and high pH. In addition, phytic acid is able to strongly chelate cations, such as Zn2+, Fe2+, Ca2+ and Mg2+ to form the phytate salts. Phytate cannot be digested by human beings or other monogastric animals due to lack of phytase, an enzyme that can hydrolyze phytate. Therefore, Zn bioavailability in seeds (or grains) is restricted by phytate. Moreover, seed Zn concentration is also reduced by elevated CO2, especially in C3 plants, such as wheat, rice and soybean. Regarding to the crucial roles but limited bioavailability of Zn, here I present a comprehensive analysis on roots, leaves (and flowering) and seeds in response to Zn deficiency in the model plant Arabidopsis thaliana via three experiments. First, I investigated the transcriptional response and whole-genome DNA methylation profile upon Zn deficiency in roots using next-generation sequencing. Ionome analysis on shoots showed that Zn concentration was strongly reduced in Zn deficiency, whereas other nutrients were not affected. Microarray Analysis identified several known Zn-deficiency responsive genes, confirming the effectiveness of Zn deficiency in this work. However, bisulfite sequencing results revealed that DNA methylation was eliminated by Zn deficiency in transposable elements and slightly in gene bodies as well. The DNA demethylation response to nutrient stress was a novel finding, as reversed to previous reports about phosphate stress which accumulated methylation. Surprisingly, further analysis suggested that DNA methylation occurred independent of gene transcription. Nevertheless, non-CpG methylation has a potential impact on flower development in response to Zn deficiency. The second experiment investigated the relationship between rosette size and flowering, and how rosette size and flowering time were regulated by Zn deficiency. Using natural variation population (168 Arabidopsis accessions), I found that flowering time was positively correlated with rosette size in early-flowering accessions but not in late-flowering accessions. Intriguingly, the flowering time was delayed by Zn deficiency in these early-flowering plants and resulting in promotion of vegetative biomass. However, Zn-regulated flowering time was independent of previously reported flowering pathways. Then genome-wide association study identified the underlying candidate gene was FLOWERING LOCUS T (FT) which was strongly inhibited by Zn deficiency in all accessions. Detailed genetic analysis confirmed this result as well. Furthermore, the promotion of leaf size in Zn deficiency was found being contributed by cell proliferation (cell number) but not cell size. Lastly, in the third experiment I was interested in the natural genetic variation in seed Zn concentration, together with iron (Fe) and manganese (Mn), in response to Zn deficiency. Across around 100 accessions, average seed Zn concentration decreased from 47.4 µg g-1 to 31.3 µg g-1 due to Zn deficiency. To identify candidate genes affecting seed Zn, Fe and Mn concentrations, genome-wide association mapping was performed. A candidate gene, inositol 1,3,4-trisphosphate 5/6-kinase 3 gene (ITPK3), was associated which is involved in phytate synthesis pathways. However, loss of this gene in itpk3-1 did neither affect phytate seed levels nor seed Zn, Fe and Mn. Nevertheless, large natural variance of micronutrient seed levels was identified in the population and several accessions maintained high seed Zn despite growth in Zn-deficient conditions. Altogether, this study presents comprehensive analyses in how Arabidopsis adapts to Zn deficiency in regard of root transcription and DNA methylation, flowering and leaf regulation, and seed mineral accumulation. I provided new possibilities of correlation between DNA methylation and gene transcription, which is much more complex than previously reported. I also opened a novel insight into flowering regulation on leaf size, resulting in promotion of vegetative biomass in nutrient deficiency. Substantial natural variation of seed experiment indicated that the evolution process was involved in seed mineral accumulation in Arabidopsis, especially those accessions maintaining Zn concentration in Zn-deficient soils are valuable for further investigations. I believe these findings in Arabidopsis also provide precious knowledge for plant breeders and agronomists who work on crops.
Dissecting the genetic basis of root- and rhizosphere-related phosphorususe efficiency in European elite maize (Zea mays L.) lines and landraces
(2021) Li, Xuelian; Ludewig, Uwe
In agriculture, farmers massively apply P fertilizer to maintain high yield. Due to the long-term high fertilization rates and long-term organic residue accumulation, the total P pool per hectare has increased between 1900 and 2020. Since modern varieties have often been selected in high-nutrient input conditions for high yields, concerns are being raised that the beneficial traits for P uptake under a limited P supply will gradually decline in elite varieties. Regarding to maize (Zea mays L.), thousands of varieties have been bred since it was domesticated as a food product. It is an open question whether traits and genes related to P deficiency in European maize have changed since the Green Revolution, the start of hybrid breeding and high-intensity fertilization. This is the core research question of this dissertation. Here I present the analysis of roots in response to P deficiency using a diverse panel of European maize genotypes via several experiments. In Chapter I, we focus on whether maize seedlings of the flint and dent heterotic pools vary in the P acquisition and utilization since the onset of hybrid breeding using 34 genotypes in mini-rhizotrons. These genotypes included 16 flint lines that were released over more than five decades ago, 7 doubled haploid lines from the flint landraces (DH_LR), 8 dent lines, and 3 hybrids. Seedling P use efficiency (PUE) and related traits were measured and compared at two P levels in a calcareous soil. In Chapter II, we compared the root exudated organic acids and mycorrhizal fungi colonization degree among 24 genotypes which have been evaluated in Chapter I. These genotypes included 16 flint lines, 6 DH_LR and 2 old dent lines. Seedling colonization with arbuscular mycorrhizal fungi (AMF) and organic acid anion release were measured. P-uptake-related root traits were compared under P-sufficient and P-deficient conditions. In Chapter III, using nearly isogenic maize lines, the B73 wild type and the rth3 root hairless mutant, we quantified the effect of root hairs and AMF infection in a calcareous soil under P deficiency. Wild-type root hairs extended the rhizosphere for acid phosphatase activity by 0.5 mm compared with the rth3 hairless mutant. Total root length of the wild type was longer than that of rth3 under P deficiency. Higher AMF colonization and mycorrhiza-induced phosphate transporter gene expression were identified in the mutant under P deficiency, but plant growth and P acquisition were similar between mutant and the wild type. The mycorrhizal dependency of maize was 33 % higher than the root hair dependency. Root hairs and AMF inoculation are two alternative ways to increase Pi acquisition under P deficiency, but these two strategies compete with each other. In Chapter IV again two nearly isogenic maize lines, the B73 wild type and the rth2 root hairless mutant, were used to address the importance of root hairs during drought and under P deficiency. The results indicate that drought and P deficiency synergistically impair maize growth; while P concentrations were little affected by the loss of root hairs, the P content was massively reduced at combined stress, showing that P deficiency is much more severe under drought. In Chapter V, we first compared the root traits response to low P and high P of six preselected genotypes in European flint in Chapter I. We then generated RNA libraries from the roots of these lines under both low P and high P. Using an expressed genes matrix, we conducted a Weighted Genomic Coexpression Network Analysis (WGCNA), and detected general low P-induced modules and modules that were higher in founder flints. The P deficiency-responsive metabolic processes common to all six genotypes included: (1) acceleration of carbon supply for organic acid synthesis through glycolysis and TCA cycle; (2) alteration of lipid metabolism; (3) changes of activity of transmembrane transporters; (4) carotenoid metabolism. Additionally, the founder flint line EP1, F2 and doubled haploid landrace SM1 have their specific strategies and mechanism to cope with low P. Our findings well support other studies with transcriptome, proteome and metabolome experiments in maize and other species, and point to molecular events involved in the efficient alleviation of P stress in efficient maize accessions. Altogether, this study presents informative analyses in how maize genotypes with distinct breeding history adapt to P deficiency in regard of root, rhizosphere traits and root transcription. It showed correlation between phenotypic traits and gene transcription, which is much more complex than previously reported. It also opened a novel insight into molecular regulation on Pi utilization, resulting in promotion of vegetative biomass in P deficiency. These findings will also provide precious knowledge for plant breeders and agronomists who work on P research in maize and other cereal crops.
Effectiveness of bio-effectors on maize, wheat and tomato performance and phosphorus acquisition from greenhouse to field scales in Europe and Israel: a meta-analysis
(2024) Nkebiwe, Peteh Mehdi; Stevens Lekfeldt, Jonas D.; Symanczik, Sarah; Thonar, Cécile; Mäder, Paul; Bar-Tal, Asher; Halpern, Moshe; Biró, Borbala; Bradáčová, Klára; Caniullan, Pedro C.; Choudhary, Krishna K.; Cozzolino, Vincenza; Di Stasio, Emilio; Dobczinski, Stefan; Geistlinger, Joerg; Lüthi, Angelika; Gómez-Muñoz, Beatriz; Kandeler, Ellen; Kolberg, Flora; Kotroczó, Zsolt; Kulhanek, Martin; Mercl, Filip; Tamir, Guy; Moradtalab, Narges; Piccolo, Alessandro; Maggio, Albino; Nassal, Dinah; Szalai, Magdolna Zita; Juhos, Katalin; Fora, Ciprian G.; Florea, Andreea; Poşta, Gheorghe; Lauer, Karl Fritz; Toth, Brigitta; Tlustoš, Pavel; Mpanga, Isaac K.; Weber, Nino; Weinmann, Markus; Yermiyahu, Uri; Magid, Jakob; Müller, Torsten; Neumann, Günter; Ludewig, Uwe; de Neergaard, Andreas
Biostimulants (Bio-effectors, BEs) comprise plant growth-promoting microorganisms and active natural substances that promote plant nutrient-acquisition, stress resilience, growth, crop quality and yield. Unfortunately, the effectiveness of BEs, particularly under field conditions, appears highly variable and poorly quantified. Using random model meta-analyses tools, we summarize the effects of 107 BE treatments on the performance of major crops, mainly conducted within the EU-funded project BIOFECTOR with a focus on phosphorus (P) nutrition, over five years. Our analyses comprised 94 controlled pot and 47 field experiments under different geoclimatic conditions, with variable stress levels across European countries and Israel. The results show an average growth/yield increase by 9.3% (n=945), with substantial differences between crops (tomato > maize > wheat) and growth conditions (controlled nursery + field (Seed germination and nursery under controlled conditions and young plants transplanted to the field) > controlled > field). Average crop growth responses were independent of BE type, P fertilizer type, soil pH and plant-available soil P (water-P, Olsen-P or Calcium acetate lactate-P). BE effectiveness profited from manure and other organic fertilizers, increasing soil pH and presence of abiotic stresses (cold, drought/heat or salinity). Systematic meta-studies based on published literature commonly face the inherent problem of publication bias where the most suspected form is the selective publication of statistically significant results. In this meta-analysis, however, the results obtained from all experiments within the project are included. Therefore, it is free of publication bias. In contrast to reviews of published literature, our unique study design is based on a common standardized protocol which applies to all experiments conducted within the project to reduce sources of variability. Based on data of crop growth, yield and P acquisition, we conclude that application of BEs can save fertilizer resources in the future, but the efficiency of BE application depends on cropping systems and environments.
Functional characterization of genes involved in development and function of cluster root in Lupinus albus
(2020) Zhou, Yaping; Ludewig, Uwe
White lupin cluster roots are specialized brush-like root structures that are formed in some species under phosphorus (P)-deficient conditions. They intensely secrete protons and organic anions for solubilization and acquisition of sparingly soluble phosphates. Because of the outstanding P efficiency of white lupin, this species has served as an illuminating model for plant adaptations to phosphorus deficiency. Since decades, numerous studies were carried out to gain a comprehensive knowledge of cluster root formation and function. The aim of this study was to identify genetic components involved in cluster root development and to identify the transporter genes that are potentially involved in citrate andmalate secretion under P deficiency as well as under toxic aluminium (Al) exposure.
Interactions of nitrogen-related, growth promoting bacteria with Miscanthus × giganteus : impact and mechanism
(2020) Liu, Yuan; Ludewig, Uwe
The highly nitrogen-use efficient biomass grass Miscanthus is a host of the bacterial endophyte Herbaspirillum frisingense. While Herbaspirillum frisingense has the genetic competence to fix nitrogen, the plant-associated microbiome may also contribute to this nitrogen efficiency. Furthermore, the costly field establishment of the sterile perennial Miscanthus × giganteus from rhizomes is a severe constraint for expanding the production area of this commercial biomass crop. In this study, the effect of Herbaspirillum frisingense inoculation on stem-cutting sprouting, shoot biomass and other yield parameters was investigated. I studied how the inoculation impacts on the M. × giganteus associated microbiome and how the long term differences in nitrogen fertilizer amount modulated the M. × giganteus associated microbiome. This was studied in a 14 year-old field trial of M. ×giganteus fertilized with various amounts of nitrogen. Stem cutting inoculation improved the shoot sprouting and establishment success of Miscanthus × giganteus in the greenhouse. In a small field trial, plant height and biomass from inoculated sites were significantly larger in the second year after establishment, but already after one year after inoculation, the bulk soil, rhizosphere, root and rhizome microbiomes were almost devoid of Herbaspirillum. This beta-proteobacterium may colonize the shoot of Miscanthus × giganteus more efficiently. Major differences between bacterial communities were determined by plant-soil compartments and less by the plant organs, while both inoculation and nitrogen had little effects on these communities. Compared to the little effect on the soil, rhizosphere and root microbiomes, the rhizome microbiome was massively modulated by both inoculation and nitrogen level. In the rhizome, several proteobacteria, which are associated with plant growth promoting functions, were enriched by inoculation, while N2-fixing-related bacterial families were favored by long-term nitrogen-deficiency plots, but denitrifier-related families were depleted. The studies suggest that H. frisingense inoculation may improve establishment of Miscanthus stem cuttings and has long-lasting effects on the rhizome microbiome diversity, despite low rhizocompetence and low root abundance. Meanwhile, the rhizome could be a potential nitrogen fixation factory. The organ-specific, nitrogen-related bacterial communities are modulated by long-term different nitrogen supply and are mainly shaped by the plant, which provides guidance for optimizing Miscanthus sustainable cultivation.
Interplay between nutrition, senescence and cytosine methylation in Arabidopsis thaliana
(2023) Vatov, Emil; Ludewig, Uwe
In monocarpic plants, senescence is the last stage of leaf development and usually leads to the death of the organism. Systematic degradation of leaf components provides nutrients for the newly developing flowers and seeds. The physiology and transcriptional changes that occur in A. thaliana during this process are very well documented. However, the involvement of epigenic mechanisms remains to be established. In this study, the role of cytosine methylation in the regulation of monocarpic leaf senescence was examined in A. thaliana. Hypomethylated ddc (drm1/2 cmt3) and hypermethylated ros1 mutants showed consistent senescence-specific phenotypes. Disrupted de-novo methylation resulted in delayed, while disrupted demethylation resulted in earlier flowering and appearance of first symptoms of senescence. Both genotypes executed the senescence program faster than Col-0, with lower leaf:seed and higher C:N ratios. During nitrogen, or phosphorus withdrawal and resupply, nutrient remobilization was not inhibited in the two mutants. However, the plant’s response in terms of changes in shoot and root growth was delayed, or non existent. Furthermore, the impact of N withdrawal on delay of the flowering time was inhibited in the two mutants. These results support involvement of cytosine methylation in stress response signaling and downstream effects on organ development and flowering times. The stress response and senescence specific phenotypes of ddc could be partially due to disrupted WRKY signaling, as loss of methylation in W-box binding sites was prevalent, specifically near the transcription start sites of ORFs, and WRKY18, 25 and 53 appeared to be sensitive towards cytosine methylation. Overall decrease in cytosine methylation levels was observed, as early as the opening of the first flowers, together with a decrease in chlorophyll concentrations and an increase in H2O2 and glucose levels in the wild type Col-0. Inhibition in maintenance methylation in the early stages of reproductive growth is consistent with these observations. A complex interaction between four cytokinins was present as early as flower induction, followed by a mass turnover of bound auxin (IAA) at flower opening, that resulted in near doubling of free IAA at seed development. Plant defense responses were induced thereafter, as an increase in salicylic acid (SA) and camalexin occurred, followed by an increase in jasmonic acid (JA) and abscisic acid (ABA). Active RNA-dependent DNA methylation (RdDM) was indicated by a moderate overrepresentation of hypermethylated CHG and CHH loci, together with partial recovery of total methylation levels at the latest stages during seed maturation. Considering the delayed senescence phenotype of ddc, de-novo methylation via RdDM appears to be involved in initiation and execution of the senescence program. Furthermore, hypomethylation at ROS1 gene regulatory region was related to down regulation of gene expression. As an antagonist of RdDM, together with the early senescence phenotype of ros1, these results strengthen the importance of de-novo methylation for senescence, while active demethylation gets down regulated. Overall, methylation changes were little related to known gene expression changes that are associated with senescence. Limited targeting of WRKY and bZIP binding sites hinders conclusions about senescence specific effects of cytosine methylation in signal transduction networks. Altogether, the present work shines light on the importance of proper maintenance of cytosine methylation for flowering time, nutrient remobilization and senescence, and identifies defined cytosine methylation changes during senescence in a comprehensive physiological framework.
The LaCLE35 peptide modifies rootlet density and length in cluster roots of white lupin
(2024) Olt, Philipp; Ding, Wenli; Schulze, Waltraud X.; Ludewig, Uwe
White lupin (lupinus albus L.) forms special bottlebrush‐like root structures called cluster roots (CR) when phosphorus is low, to remobilise sparingly soluble phosphates in the soil. The molecular mechanisms that control the CR formation remain unknown. Root development in other plants is regulated by CLE (CLAVATA3/EMBRYO SURROUNDING REGION (ESR)‐RELATED) peptides, which provide more precise control mechanisms than common phytohormones. This makes these peptides interesting candidates to be involved in CR formation, where fine tuning to environmental factors is required. In this study we present an analysis of CLE peptides in white lupin. The peptides LaCLE35 (RGVHyPSGANPLHN) and LaCLE55 (RRVHyPSCHyPDPLHN) reduced root growth and altered CR in hydroponically cultured white lupins. We demonstrate that rootlet density and rootlet length were locally, but not systemically, impaired by exogenously applied CLE35. The peptide was identified in the xylem sap. The inhibitory effect of CLE35 on root growth was attributed to arrested cell elongation in root tips. Taken together, CLE peptides affect both rootlet density and rootlet length, which are two critical factors for CR formation, and may be involved in fine tuning this peculiar root structure that is present in a few crops and many Proteaceae species, under low phosphorus availability.
Loss of LaMATE impairs isoflavonoid release from cluster roots of phosphorus‐deficient white lupin
(2021) Zhou, Yaping; Olt, Philipp; Neuhäuser, Benjamin; Moradtalab, Narges; Bautista, William; Uhde‐Stone, Claudia; Neumann, Günter; Ludewig, Uwe
White lupin (Lupinus albus L.) forms brush‐like root structures called cluster roots under phosphorus‐deficient conditions. Clusters secrete citrate and other organic compounds to mobilize sparingly soluble soil phosphates. In the context of aluminum toxicity tolerance mechanisms in other species, citrate is released via a subgroup of MATE/DTX proteins (multidrug and toxic compound extrusion/detoxification). White lupin contains 56 MATE/DTX genes. Many of these are closely related to gene orthologs with known substrates in other species. LaMATE is a marker gene for functional, mature clusters and is, together with its close homolog LaMATE3, a candidate for the citrate release. Both were highest expressed in mature clusters and when expressed in oocytes, induced inward‐rectifying currents that were likely carried by endogenous channels. No citrate efflux was associated with LaMATE and LaMATE3 expression in oocytes. Furthermore, citrate secretion was largely unaffected in P‐deficient composite mutant plants with genome‐edited or RNAi‐silenced LaMATE in roots. Moderately lower concentrations of citrate and malate in the root tissue and consequently less organic acid anion secretion and lower malate in the xylem sap were identified. Interestingly, however, less genistein was consistently found in mutant exudates, opening the possibility that LaMATE is involved in isoflavonoid release.
Managing crop health by mineral nitrogen fertilization and use of different chemical nitrogen forms
(2023) Maywald, Niels Julian; Ludewig, Uwe
Maintaining plant health is one of the most difficult but crucial challenges in crop production to realize plants’ full genetic potential. It is lowered by a variety of abiotic and biotic stresses that are becoming more severe and unpredictable due to climate change and its consequences. In addition, the use of chemical synthetic pesticides is increasingly criticized for endangering sensitive natural resources and possible pesticide residues in food and environment. Avoiding or reducing the use of chemical synthetic plant protection products makes the control of phytopathogenic pests even more difficult. Therefore, in addition to optimizing various management measures such as tillage, sowing time, row spacing or crop rotation, mineral nitrogen (N) fertilization and the targeted application of N forms must be utilized to reduce abiotic stress factors and the infestation pressure of certain pests to ensure high yield performance. Consequently, several experiments were conducted to better understand how mineral nitrogen fertilization and forms can improve plant health by increasing plant resistance to abiotic stressors, particularly repeated drought stress and nutrient (P) deficiency, and to biotic stressors, such as relevant phytopathogenic fungi. It was found that with respect to repeated drought stress, maize plants receiving supplemental nitrogen during the recovery period after an early drought stress were better able to cope with late drought stress. In this context, N fertilization could help the plant to maintain its photosynthetic activity under drought stress. Additionally, plants repeatedly exposed to drought stress recovered faster with N fertilization due to transiently higher antioxidant levels and higher production of reactive oxygen species. A further experiment revealed that depending on the maize genotype, ammonium as a form of nitrogen has a positive effect on the availability and uptake of phosphorus compared to nitrate, depending on the maize genotype. This observation could be attributed not only to the acidifying effect on the pH of the rhizosphere, but also to the increased abundance of various phosphorus-solubilizing bacteria and arbuscular mycorrhizal fungi under ammonium nutrition. Together this could provide an enhanced P availability, which ultimately reduces plant stress and improves physiologically resistance leading to a reduction in disease risk. Nevertheless, studies revealed that high N fertilization in most cases promotes disease attack and makes the plant more susceptible to pathogens. Scrutinization of this observation indicated that N fertilization enhances infestations of biotrophic pathogens, especially in wheat, while necrotrophic fungi were attenuated. Overall, the complex relationship between plant pathogens and nitrogen nutrition appears to be highly variable due to dynamic factors such as the soil, microorganisms in the rhizosphere, environmental factors, and the host plant, making it difficult to give definite statements about the effects of nitrogen nutrition on pathogen occurrence. Thus, the form of nitrogen could be a promising way to target nitrogen fertilization against individual pathogens. With regards to the previous research, experiments on the influence of N form on pathogen infection, revealed that wheat leaves inoculated with the foliar pathogen Blumeria graminis f. sp. tritici (Bgt) were comparatively less infested when fertilized with nitrate or cyanamide compared to ammonium. After contact with the pathogen, an enhanced defense response in form of increased production of protective substances, indicated by increased concentrations of hydrogen peroxide and superoxide dismutase, and increased antioxidant potential, was detected. Further, it was observed that ammonium fertilization resulted in lower bacterial richness in the plant rhizosphere and higher fungal richness compared to nitrate supplementation. Additionally, a pronounced effect of ammonium fertilization on rootcolonization by important fungal pathogens such as Gaeumannomyces graminis var. tritici (Ggt) and Bgt was found. Regarding the experiment with maize under low P conditions, it appears that ammonium is able to promote both pathogenic and beneficial fungi in cereal crops. Thus, nitrate fertilization appears not only to suppress the occurrence of fungi, but may also promote pathogen-antagonistic bacteria, which in turn have a positive effect on fungal disease suppression.
Microbial consortia versus single-strain inoculants as drought stress protectants in potato affected by the form of N supply
(2024) Mamun, Abdullah Al; Neumann, Günter; Moradtalab, Narges; Ahmed, Aneesh; Dupuis, Brice; Darbon, Geoffrey; Nawaz, Fahim; Declerck, Stephane; Mai, Karin; Vogt, Wolfgang; Ludewig, Uwe; Weinmann, Markus
This study investigated the drought protection effects of six fungal and bacterial inoculants and ten consortia thereof on vegetative growth, nutritional status, and tuberization of potato under controlled and field conditions. It was hypothesized that microbial consortia offer improved drought protection as compared with single strains, due to complementary or synergistic effects, with differential impacts also of N fertilization management. Under NO3− fertilization, a 70% reduction in water supply over six weeks reduced shoot and tuber biomass of non-inoculated plants by 30% and 50%, respectively, and induced phosphate (P) limitation compared to the well-watered control. The P nutritional status was significantly increased above the deficiency threshold by three single-strain inoculants and eight consortia. This was associated with the presence of the arbuscular mycorrhizal fungus (AMF) inoculant Rhizophagus irregularis MUCL41833 (five cases) and stimulation of root growth (five cases). Additionally, Bacillus amyloliquefaciens FZB42 and AMF + Pseudomonas brassicacearum 3Re2-7 significantly reduced irreversible drought-induced leaf damage after recovery to well-watered conditions. However, the microbial inoculants did not mitigate drought-induced reductions in tuber biomass, neither in greenhouse nor in field experiments. By contrast, NH4+-dominated fertilization significantly increased tuber biomass under drought stress (534%), which was further increased by additional AMF inoculation (951%). This coincided with (i) improved enzymatic detoxification of drought-induced reactive oxygen species (ROS), (ii) improved osmotic adjustment in the shoot tissue (glycine betaine accumulation), (iii) increased shoot concentrations of ABA, jasmonic acid, and indole acetic acid, involved in drought stress signaling and tuberization, and (iv) reduced irreversible drought-induced leaf damage. Additional application of bacterial inoculants further improved ROS detoxification by increasing the production of antioxidants but stimulated biomass allocation towards shoot growth at the expense of tuber development. The results demonstrated that microbial consortia could increase the probability of drought protection effects influenced by the form of N supply. However, protective effects on vegetative growth do not necessarily translate into yield benefits, which can be achieved by adequate combination of inoculants and fertilizers.
Molecular regulation of components of root development and nutrient uptake in white lupin (Lupinus albus L.)
(2023) Olt, Philipp; Ludewig, Uwe
White lupin (Lupinus albus L.) is specially adapted to sites with low availability of plant-available phosphorus (P), which is of particular agricultural importance because of chemical P fixation in the soil and limited reserves of P fertilizer resources. With special root structures, the cluster roots (syn. proteoid roots), it considerably increases the root surface area and excretes root exudates such as anions of organic acids, which make it possible to release phosphate ions from poorly soluble phosphate compounds in the soil and make them available to plants. The characteristic structure of these cluster roots usually consists of a lateral root with certain sections that have a significantly higher density of further lateral roots (rootlets) than the rest of the lateral root. In addition, the rootlets are evenly limited in length and, as they grow bundled in the cluster sections, the structure of cluster roots is reminiscent of a bottle brush. Cluster roots are also formed by other plant species such as some species from the Proteaceae family, but in contrast to these slow- growing and perennial woody plants, white lupin with its short life cycle and small size is an ideal model organism for the study of these special root structures. In addition to the mechanisms involved in the function of cluster roots, the regulation of formation and development of cluster roots is also of great importance for basic research in this field. Three studies were carried out as part of this thesis to examine these aspects in more detail. In order to better understand the functional mechanisms involved in the excretion of exudates, the hypothesis that the METAL AND TOXIN EXTRUSION (MATE) transport proteins LaMATE and LaMATE3 transport citrate was tested in the first study. The similarity of the gene sequences of these white lupin proteins with proteins from the MATE/DTX family, of which citrate transport is already known, as well as the increased gene expression of LaMATE and LaMATE3 in mature cluster roots led to this assumption. However, electrophysiological studies of the proteins with 13C- labeled citrate showed that LaMATE and LaMATE3 probably do not transport citrate and also the analysis of root exudates from transient loss-of-function mutants could not confirm the involvement of LaMATE in the transport of citrate. However, the excretion of the isoflavonoid genistein was found to be significantly reduced in the transient loss-of-function mutants, leading to the hypothesis that LaMATE may be involved in the exudation of isoflavonoids in mature cluster roots. As a result of the mobilization of phosphates through the excretion of organic acids, other cations such as manganese (Mn) also dissolve, which leads to increased Mn concentrations in the soil solution. As manganese uptake in the roots is not actively regulated, Mn accumulates in the plant, which has a toxic effect in higher concentrations. For this reason, white lupin needs a strategy to counteract toxic manganese accumulation, which was investigated in more detail in the second study. The observation that a greater increase in Mn concentration could be measured in the leaves than in the roots after elevated Mn exposure of white lupin indicates an actively regulated transport of excess Mn in the plant. The METAL TOLERANCE PROTEIN (MTP) AtMTP8 is involved in the detoxification of excess Mn in Arabidopsis and the increased gene expression of the corresponding white lupin homolog LaMTP8.1 in plants exposed to elevated Mn concentration suggested that LaMTP8.1 also fulfills a detoxification function. In further experiments, the ability of the LaMTP8.1 protein to transport Mn was demonstrated by heterologous expression of LaMTP8.1 in yeast cells. Furthermore, the high Mn concentrations in the leaves already indicated that the sink of Mn sequestration is located there and since AtMTP8 transports Mn into the vacuole, it was assumed that LaMTP8.1 could be localized in the tonoplast of the leaf cells to transport excess Mn into the vacuoles. This hypothesis was confirmed by homologous expression of LaMTP8.1 combined with a fluorescent marker in white lupin protoplasts. In summary, this study demonstrated that LaMTP8.1 is a vacuolar Mn transporter that mediates the transport of Mn into the vacuoles of leaf cells to detoxify excess Mn. While the first two studies addressed functional and physiological aspects of cluster roots, the third study focused on the mechanisms of formation and development of these root structures. To this end, the main focus was on the CLAVATA3/ EMBRYO SURROUNDING REGION (ESR)- RELATED (CLE) peptide family, of which some members regulate root growth in other plant species and enable more precise control of regulation compared to the known growth regulators auxin and cytokinin. In a comprehensive analysis, 30 known and further 25 new, putative CLE peptides were identified in Lupinus albus. Several of the CLE peptides were tested in a hydroponic system on young white lupins for their effects on root development and cluster root formation. The two CLE peptides LaCLE35 (RGVHyPSGANPLHN) and LaCLE55 (RRVHyPSCHyPDPLHN) showed striking inhibitory effects and altered both root growth and cluster root development in an inhibitory manner. The peptide LaCLE35 stood out in particular because it was the only CLE peptide detected in white lupin xylem sap and was therefore investigated in more detail. It was shown that LaCLE35 influences both the density and the length of cluster rootlets, and thus has an effect on the two crucial factors of cluster root formation. The inhibitory effect of CLE35 could be attributed to a suppression of cell elongation and further experiments with split-root setups showed that the externally added synthetic peptide LaCLE35 has a local but not a systemic effect. The investigations of the LaMATE transport proteins and LaMTP8.1-mediated Mn detoxification as well as the overview of the detected CLE peptides in white lupin and the analysis of the inhibitory influences of LaCLE35 on cluster roots form the basis of this thesis and aim to contribute to the understanding of the function, effects and formation of these special root structures.
Nitrogen improves the recovery of maize plants under repeated drought stress
(2022) Maywald, Niels Julian; Hernández‐Pridybailo, Andrés; Ludewig, Uwe
Background Modern high-yielding crops, such as maize, are characterized by extensive yield stability across various environments and can cope with repetitive periods of moderate water shortage. However, there is conflicting evidence on how the nutritional status of the plants contributes to stress resilience and whether farmers have management options via nitrogen fertilization. Aims We aimed at identifying factors relevant for improved growth recovery of maize after repeated water deficit stress (WDS). Methods A pot experiment with maize and repeated WDS was conducted. Growth and recovery from stress and physiological parameters were measured. Results The growth penalty of juvenile maize plants exposed to a moderate WDS was lost after additional exposure to a 2-week WDS. Primed plants transiently contained more osmolytes and performed superior in the second recovery phase when nitrogen fertilization was applied directly before the second WDS. Nitrogen fertilization did not affect the osmolyte quantity, and primed plants had transiently higher antioxidant levels, higher reactive oxygen species production and recovered more quickly with N addition. Conclusions Pot experiments suggest that nitrogen fertilization may be an option to improve maize resilience to repeated WDS, a hypothesis that should be tested more rigorously in the field.
Nutritional regulation of DNA methylation and gene expression in maize
(2018) Mager, Svenja; Ludewig, Uwe
DNA methylation in plants plays a role in transposon silencing, genome stability and gene expression regulation. Environmental factors alter the methylation pattern of DNA and recently nutrient stresses, such as phosphate starvation, were shown to alter DNA methylation. DNA methylation had been frequently addressed in plants with notably small genomes that are poor in transposons. Here, part of the DNA methylome of nitrogen-, phosphorus- and zinc-deficient (-N, -P and -Zn, respectively) maize roots were compared by reduced representation sequencing and their relationship with gene expression under prolonged stresses analyzed. Tremendous DNA methylation loss was encountered in maize under nitrogen and zinc deficiency, but much less under phosphorus deficiency. This occurred only in the symmetrical cytosine contexts, predominantly in CG context, but also in the CHG context. In contrast to other plants, differential methylation in the more flexible CHH context was essentially absent. For each sample, specific nutrient deficiency-regulated genes were differentially expressed. In -Zn samples the lowest number of differentially expressed genes was found while -N and -P samples contained a similar number of differentially expressed genes. For all samples, differentially methylated regions (DMRs) were predominantly identified in transposable elements (TEs). A minor fraction of such DMRs was associated with altered gene expression of nearby genes in -N and -P. Interestingly, although these TEs were mostly hypomethylated, they were associated with both upand down-regulated gene expression. For -Zn, these associations were not found but a correlation between hypomethylation of gene bodies and expression of some genes. Here again, hypomethylation occurred with up- and downregulation of gene expression. The results suggested a different methylome regulation in maize compared to rice and Arabidopsis upon nutrient deficiencies indicating a nutrient- and species-specific association of genomic DNA methylation and gene expression. The limited correlation between differential DNA methylation and gene expression suggested that heritable regulation of the expression of nutrient deficiency-regulated genes was not the primary function of the methylation loss. Rather, the major function of the DNA methylation loss in this experiment may have been to increase the genetic diversity in the next generation by increased frequency of recombination events, mutations and transposable element movements.
Physiological, molecular, and epigenetic aspects of early transient nitrogen deprivation recovery in maize
(2024) Hernandez Pridybailo, Andres; Ludewig, Uwe
Maize is a widely cultivated crop and a primary food source for humans and livestock. Along with its substantial contribution to biomass for fuel production, maize is the most produced cereal globally. However, despite its prevalence, improving nitrogen (N) use efficiency in maize presents ongoing challenges. Understanding how maize plants utilize nitrogen is crucial for identifying traits that could aid breeders in addressing this efficiency gap. When plants experience transient stress and return to previous conditions, a recovery phase is initiated, acclimating the plants and potentially enhancing their responses to subsequent stress events. This mild or transient stress is termed "eustress," and its intentional manipulation is referred to as "crop priming." Crop priming, extensively studied in the context of drought, was explored in our previous research, which revealed that nitrogen supplementation during recovery from early transient water deficits enhances priming effects. This highlights the pivotal role of N in priming and that impaired N supply might negatively affect any acclimation to future stresses. Notably, there is limited literature on transient N deprivation, with most studies focusing on plant responses under conditions of maintained N deficiency. Furthermore, epigenetic regulation of plant mineral nutritional responses via DNA methylation has been reported, like many aspects of plant development that are regulated by such mechanism. Epigenetics is the study of phenotypic changes that can be inherited through mitosis or meiosis, which cannot be explained by changes in the DNA sequence. DNA methylation is the covalent modification of cytosines by the addition of a methyl group, which, depending on its location in the genome, can affect genomic stability and gene expression. Previous results have revealed that N deficiency modifies the methylome of maize roots, hypomethylating transposable elements in a nutrient-specific way. Weak correlations between DNA methylation and gene expression were observed, but a deeper insight into how this covalent modification of cytosines is related to plant mineral nutrition is lacking. DNA methylation patterns are heritable in a sort of “epigenetic memory” and may arise after an environmental stimulus. Here, the role of DNA methylation and its relationship with physiological responses during transient N deprivation were studied. The Fast-Flowering Mini-Maize inbred line A (FFMM-A) was chosen for this study because it can be easily grown hydroponically under controlled conditions. Maize ear leaves recovering from initial nitrogen deprivation (without water limitation) exhibited tissue-specific differences in rapidly dividing meristematic and mature photosynthetically active tissues. Through a series of experiments, a 60 h N deprivation period at the V1 stage was established as a nutritional stress that allowed plants to show mild N deprivation symptoms from which the plants fully recovered. Kinematic analysis of the recovering ear leaf revealed a slight decrease in leaf blade length due to reduced leaf meristem size and leaf number, resulting in a reduced cell production rate. Interestingly, the leaf elongation rate (LER) was maintained, causing a higher specific leaf area, which affected the leaf blade structure. Transcriptomic analysis of the cell division and maturation zones of the recovering ear leaf showed altered gene expression related to cell cycle, lipid metabolism, and secondary metabolism, including phytohormone regulation. Notably, cell cycle-related proteins are also involved in DNA repair and may be involved in somatic memory effects. If plants that had been exposed to early transient N-deficiency stress were later subjected to a second N-deprivation stress at the V5 stage, physiological measurements showed that early N deprivation affected upper leaf chlorophyll accumulation during late N-deprivation recovery. These findings suggest that early N deprivation has long-term effects, especially in dividing tissues, from which young and mature tissues are produced by replication. Furthermore, I identified that the loss of DNA methylation makes maize plants more susceptible to early N deprivation. Loss-of-function zmet2 allele was used in this study. Examination of the methylome of the zmet2 and zmet5 mutants provided insights into the relationship between DNA methylation patterns, nitrogen responses, and the expression of developmentally regulated genes in the leaf. These alleles correspond to CHROMOMETHYLASE3-like maintenance methyltransferase genes, and their mutation causes generalized genome hypomethylation, mostly in the CHG and CHH contexts. After a 96 h N deprivation period at the V1 stage, the zmet2 mutants failed to modify their root-to-shoot ratio and to maintain the LER, with respect to the isogenic B73 line. Moreover, machine learning identified interesting clusters according to their expression patterns from the leaf transcriptome of the dividing and maturation zones of FFMM-A plants. Reanalysis of publicly available datasets from B73 seedling leaf methylation patterns and those of the zmet2 and zmet5 mutants was used to compare the expression patterns of the gene clusters, their methylation patterns, and selected genome features. Interestingly, the methylation pattern of the gene body was highly correlated with the level of expression in the dividing zone of the developing ear leaf. Collectively, mutants in the methylation maintenance pathway, rather than being susceptible to N deprivation, failed to develop symptoms of recovery from such stress, which might be related to the regulation of N metabolism-related genes by DNA methylation. Additionally, the closely related gene expression pattern in the dividing zone and intron non-CpG methylation suggests an intimate role of maintenance of DNA methylation in N nutrition.
Plant ammonium transporter (AMT) integration in regulatory networks
(2016) Straub, Tatsiana; Ludewig, Uwe
Ammonium is a ubiquitous key nutrient in agricultural soils and the preferred nitrogen source for plants. However, excessive ammonium accumulation represses plant growth and development. Ammonium is taken up by plant cells via high-affinity ammonium transporters (AMTs). Six AMT genes were identified in Arabidopsis, which are separated in two distinct clades, five AMT1s and one AMT2. In the plasma membrane, AMT proteins form homo- and heterotrimers with extra-cytoplasmic N-termini and cytoplasmic C-termini. In addition to transcriptional and post-transcriptional control of AMTs by ammonium, phosphorylation in the C-terminus serves as a rapid allosteric switch of the AMT activity and prevents further internal ammonium accumulation. In a physiological screen, a kinase (CIPK23) was identified, which directly regulates ammonium transport activity under high-NH4+ conditions. Interestingly, CIPK23 is already known to regulate nitrate and potassium uptake in roots. Lesion of the CIPK23 gene significantly increased ammonium uptake, but caused growth inhibition. As expected, cipk23 plants were also limited in potassium accumulation, but high potassium availability failed to rescue the cipk23 phenotype. Furthermore, cipk23 plants were more susceptible to methylammonium (MeA), a non-metabolizable analogue of ammonium. The sensitivity to MeA was lost upon genetic suppression of AMT1 genes in the cipk23 background. The data suggest that CIPK23 directly phosphorylates AMT1s in a complex with CBL1 (calcineurin B-like protein) and thereby regulates transport activity. The expression of the CIPK23 and the CBL1 genes were ammonium-dependent and increased when N-starved plants were resupplied with ammonium. Furthermore, cbl1 mutants had enhanced NH4+ accumulation; this phenocopies the larger ammonium uptake in the cipk23 loss-of-function mutant. In vivo experiments demonstrated bimolecular interaction between CIPK23, AMT1;1, and AMT1;2, but not with AMT2;1, suggesting direct phosphorylation of AMT1-type ammonium transporters by CIPK23. However, Western blot analysis with the cipk23 mutant suggested that the loss of the kinase was not sufficient to completely abolish AMT1;1 and AMT1;2 phosphorylation, indicating several independent pathways to regulate ammonium transport activity in AMT trimers. The data identify complex post-translational regulation of ammonium transporters via the CBL1–CIPK23 pathway, which ensures reduction of AMT1 activity and suppression of ammonium uptake under high external NH4+ concentrations.
Regulation of ammonium transport in Arabidopsis thaliana
(2022) Ganz, Pascal; Ludewig, Uwe
The overarching question of this thesis deals with how plants ensure the selective uptake of ammonium while maintaining ion and pH homeostasis. A key component of this is ammonium transporters (AMTs) with high affinity towards their substrate, which are at the same time part of a multilayered protection system against uncontrolled ammonium influx. Conserved protein sequences inside the transporter were analyzed as well as the regulatory system based on post-translational modification of the transporter.
Role of benzoic acid and lettucenin A in the defense response of lettuce against soil-borne pathogens
(2021) Windisch, Saskia; Walter, Anja; Moradtalab, Narges; Walker, Frank; Höglinger, Birgit; El-Hasan, Abbas; Ludewig, Uwe; Neumann, Günter; Grosch, Rita
Soil-borne pathogens can severely limit plant productivity. Induced defense responses are plant strategies to counteract pathogen-related damage and yield loss. In this study, we hypothesized that benzoic acid and lettucenin A are involved as defense compounds against Rhizoctonia solani and Olpidium virulentus in lettuce. To address this hypothesis, we conducted growth chamber experiments using hydroponics, peat culture substrate and soil culture in pots and minirhizotrons. Benzoic acid was identified as root exudate released from lettuce plants upon pathogen infection, with pre-accumulation of benzoic acid esters in the root tissue. The amounts were sufficient to inhibit hyphal growth of R. solani in vitro (30%), to mitigate growth retardation (51%) and damage of fine roots (130%) in lettuce plants caused by R. solani, but were not able to overcome plant growth suppression induced by Olpidium infection. Additionally, lettucenin A was identified as major phytoalexin, with local accumulation in affected plant tissues upon infection with pathogens or chemical elicitation (CuSO4) and detected in trace amounts in root exudates. The results suggest a two-stage defense mechanism with pathogen-induced benzoic acid exudation initially located in the rhizosphere followed by accumulation of lettucenin A locally restricted to affected root and leaf tissues.
Site-dependent differences in DNA methylation and their impact on plant establishment in Populus trichocarpa
(2016) Schönberger, Brigitte; Ludewig, Uwe
Phosphate (Pi) limits total biomass production in natural tree ecosystems. Due to the low mobility of Pi in soil, higher plants, like trees, require special adaptations for phosphorus (P) acquisition. The genetic and physiological basis of this adaptation has been studied extensively. In addition, phosphorus starvation was recently suggested to affect epigenetic modifications in varying annual plant species. However, the impact of differential DNA methylation and microRNAs (miRNAs) on gene expression as well as site-dependent P-related physiology is largely unknown in perennials. In this study Populus trichocarpa clones, established from stem cuttings from two different locations, were grown in hydroponic culture with different P levels. Morphological and physiological parameters as well as, using bisulfite sequencing, site-specific genome-wide methylomes were determined. Gene and miRNA expression of differentially methylated regions was quantified via qPCR. Site-dependent differences in plant establishment were encountered, together with site-specific differentially methylated chromosomal regions. Methylation differences were nucleotide context-specific and extensively regulated miRNAs and their target genes in an organ-specific way. Though no direct relation between differential methylation in coding regions and their corresponding gene expression was observed, a general site-dependent transcriptional repression by DNA methylation was detected. Nevertheless, differential DNA methylation and gene expression was not affected by P nutrition, although recent studies described P-starvation induced DNA methylation changes, suggesting species-specific epigenetic mechanisms. However, differentially methylated miRNAs, together with their target genes, showed P-dependent expression profiles, indicating miRNA expression changes as a P-related epigenetic modification in poplar. Hence, it was shown that differences in DNA methylation or differentially methylated miRNAs might influence plant establishment and partially correlate with P acquisition, and thus be responsible for a site-dependent adaptation and growth performance, interesting for plant breeding, conservation biology and biodiversity studies of vegetatively propagated perennials.