Environmental genetics of root system architecture

The root system is the plant’s principal organ for water and mineral nutrient supply. Root growth follows an endogenous, developmental programme. Yet, this programme can be modulated by external cues which makes root system architecture (RSA), the spatial configuration of all root parts, a highly plastic trait. Presence or absence of nutrients such as nitrate (N), phosphate (P), potassium (K) and sulphate (S) serve as environmental signals to which a plant responds with targeted proliferation or restriction of main or lateral root growth. In turn, RSA serves as a quantitative reporter system of nutrient starvation responses and can therefore be used to study nutrient sensing and signalling mechanisms. In this study, I have analysed root architectural responses of various Arabidopsis thaliana genotypes (wildtype, mutants and natural accessions) to single and multiple nutrient deficiency treatments. A comprehensive analysis of combinatorial N, P, K an S supply allowed me to dissect the effect of individual nutrients on individual root parameters. It also highlighted the existence of interactive effects arising from simultaneous environmental stimuli. Quantification of appropriate RSA parameters allowed for targeted testing of known regulatory genes in specific nutritional settings. This revealed, for example, a novel role for CIPK23, AKT1 and NRT1.1 in integrating K and N effects on higher order lateral root branching and main root angle. A significant contribution to phenotypic variation also arose from P*K interactions. I could show that the iron (Fe) concentration in the external medium is an important driving force of RSA responses to low-P and low-K. In fact, P and K deprivation caused Fe accumulation in distinct parts of the root system, as demonstrated by Fe staining and synchrotron X-Ray fluorescence. Again, selected K, P and Fe transport and signalling mutants were tested for aberrant low-K and/or low-P phenotypes. Most notably, the two paralogous ER-localised multicopper oxidases LPR1 and LPR2 emerged as important signalling components of P and K deprivation, potentially integrating Fe homeostasis with meristematic activity under these conditions. In addition to the targeted characterisation of specific genotype-environment interactions, I investigated novel RSA responses to low-K via a non-targeted approach based on natural variation. A morphological gradient spanned the entire genotype set, linking two extreme strategies of low-K responses. Strategy I accessions responded to low-K with a moderate reduction of main root growth but a severe restriction of lateral root elongation. In contrast, strategy II genotypes ceded main root growth in favour of lateral root proliferation. The genetic basis of these low-K responses was then subsequently mapped onto the A. thaliana genome via quantitative trait loci (QTL) analysis using recombinant inbred lines derived from parental accessions that either adopt strategy I (Col-0) or II (Ct-1). In sum, this study addresses the question how plants incorporate environmental signals to modulate developmental programmes that underly RSA formation. I present evidence for novel phenotypic responses to nutrient deprivation and for novel genetic regulators involved in nutrient signalling and crosstalk

Analysis of the root system architecture of Arabidopsis provides a quantitative readout of crosstalk between nutritional signals

As plant roots forage the soil for food and water, they translate a multifactorial input of environmental stimuli into a multifactorial developmental output that manifests itself as root system architecture (RSA). Our current understanding of the underlying regulatory network is limited because root responses have traditionally been studied separately for individual nutrient deficiencies. In this study, we quantified 13 RSA parameters of Arabidopsis thaliana in 32 binary combinations of N, P, K, S, and light. Analysis of variance showed that each RSA parameter was determined by a typical pattern of environmental signals and their interactions. P caused the most important single-nutrient effects, while N-effects were strongly light dependent. Effects of K and S occurred mostly through nutrient interactions in paired or multiple combinations. Several RSA parameters were selected for further analysis through mutant phenotyping, which revealed combinations of transporters, receptors, and kinases acting as signaling modules in K–N interactions. Furthermore, nutrient response profiles of individual RSA features across NPK combinations could be assigned to transcriptionally coregulated clusters of nutrient-responsive genes in the roots and to ionome patterns in the shoots. The obtained data set provides a quantitative basis for understanding how plants integrate multiple nutritional stimuli into complex developmental programs

Diffusion and Precipitation Processes in Iron-Based Silica Gardens

Silica gardens are tubular structures that form along the interface of multivalent metal salts and alk. solns. of sodium silicate, driven by a complex interplay of osmotic and buoyant forces together with chem. reaction. They display peculiar plant-​like morphologies and thus can be considered as one of the few examples for the spontaneous biomimetic self-​ordering of purely inorg. materials. Recently, we could show that silica gardens moreover are highly dynamic systems that remain far from equil. for considerable periods of time long after macroscopic growth is completed. Due to initial compartmentalization, drastic concn. gradients were found to exist across the tube walls, which give rise to noticeable electrochem. potential differences and decay only slowly in a series of coupled diffusion and pptn. processes. The effect of the nature of the metal cations on the dynamic behavior of the system has been studied. The authors have grown single macroscopic silica garden tubes by controlled addn. of sodium silicate sol to pellets of iron(II) and iron(III) chloride. In the following, the concns. of ionic species were measured as a function of time on both sides of the formed membranes, while electrochem. potentials and pH were monitored online by immersing the corresponding sensors into the two sepd. soln. reservoirs. At the end of the expts., the solid tube material was furthermore characterized with respect to compn. and microstructure by a combination of ex situ techniques. The collected data are compared to the previously reported case of cobalt-​based silica gardens and used to shed light on ion diffusion through the inorg. membranes as well as progressive mineralization at both surfaces of the tube walls. These results reveal important differences in the dynamics of the three studied systems, which can be explained based on the acidity of the metal cations and the porosity of the membranes, leading to substantially dissimilar time-​dependent soln. chem. as well as distinct final mineral structures. The insight gained in this work may help to better understand the diffusion properties and pptn. patterns in tubular iron (hydr)​oxide​/silicate structures obsd. in geol. environments and during steel corrosion

Isotope Ratio Outlier Analysis (IROA) for HPLC–TOFMS-Based Metabolomics of Human Urine

Metabolic fingerprinting by mass spectrometry aims at the comprehensive, semiquantitative analysis of metabolites. Isotope dilution, if successfully implemented, may provide a more reliable, relative quantification. Therefore, the 13C labeled yeast extract of the IROA TruQuant kit was added as an internal standard (IS) to human urine samples measured in full-scan mode on a high-performance liquid chromatography-time-of-flight mass spectrometer (HPLC–TOFMS) system. The isotope ratio approach enabled the analysis of 112 metabolites. The correlation with reference data did not improve significantly using 12C/13C ratios compared to absolute 12C peak areas. Moreover, using an intricate 13C-labeled standard increased the complexity of the mass spectra, which made correct signal annotation more challenging. On the positive side, the ratio approach helps to reduce batch effects, but it does not perform better than computational methods such as the “removebatcheffect” function in the R package Limma

Precipitation and Crystallization Kinetics in Silica Gardens

Silica gardens are extraordinary plant-like structures resulting from the complex interplay of relatively simple inorganic components. Recent work has highlighted that macroscopic self-assembly is accompanied by the spontaneous formation of considerable chemical gradients, which induce a cascade of coupled dissolution, diffusion, and precipitation processes occurring over timescales as long as several days. In the present study, this dynamic behavior was investigated for silica gardens based on iron and cobalt chloride by means of two synchrotron- based techniques, which allow the determination of concentration profiles and time-resolved monitoring of diffraction patterns, thus giving direct insight into the progress of dissolution and crystallization phenomena in the system. On the basis of the collected data, a kinetic model is proposed to describe the relevant reactions on a fundamental physicochemical level. The results show that the choice of the metal cations (as well as their counterions) is crucial for the development of silica gardens in both the short and long term (i. e. during tube formation and upon subsequent slow equilibration), and provide important clues for understanding the properties of related structures in geochemical and industrial environments

Divergent effects of itaconate isomers on Coxiella burnetii growth in macrophages and in axenic culture

Aconitate decarboxylase-1 (ACOD1) is expressed by activated macrophages and generates itaconate that exerts anti-microbial and immunoregulatory effects. ACOD1-itaconate is essential for macrophage-mediated control of the intracellular pathogen Coxiella (C.) burnetii, which causes Q fever. Two isomers of itaconate, mesaconate and citraconate, have overlapping yet distinct activity on macrophage metabolism and inflammatory gene expression. Here, we found that all three isomers inhibited the growth of C. burnetii in axenic culture in ACCM-2 medium. However, only itaconate reduced C. burnetii replication efficiently in Acod1-/- macrophages. In contrast, addition of citraconate strongly increased C. burnetii replication in Acod1+/- macrophages, whereas mesaconate weakly enhanced bacterial burden in Acod1-/- macrophages. Analysis of intracellular isomers showed that exogenous citraconate and mesaconate inhibited the generation of itaconate by infected Acod1+/- macrophages. Uptake of added isomers into Acod1-/- macrophages was increased after infection for itaconate and mesaconate, but not for citraconate. Mesaconate, but not citraconate, competed with itaconate for uptake into macrophages. Taken together, inhibition of itaconate generation by macrophages and interference with the uptake of extracellular itaconate could be identified as potential mechanisms behind the divergent effects of citraconate and mesaconate on C. burnetii replication in macrophages or in axenic culture

MCT4 blockade increases the efficacy of immune checkpoint blockade

Background & Aims Intratumoral lactate accumulation and acidosis impair T-cell function and antitumor immunity. Interestingly, expression of the lactate transporter monocarboxylate transporter (MCT) 4, but not MCT1, turned out to be prognostic for the survival of patients with rectal cancer, indicating that single MCT4 blockade might be a promising strategy to overcome glycolysis-related therapy resistance. Methods To determine whether blockade of MCT4 alone is sufficient to improve the efficacy of immune checkpoint blockade (ICB) therapy, we examined the effects of the selective MCT1 inhibitor AZD3965 and a novel MCT4 inhibitor in a colorectal carcinoma (CRC) tumor spheroid model co-cultured with blood leukocytes in vitro and the MC38 murine CRC model in vivo in combination with an antibody against programmed cell death ligand-1(PD-L1). Results Inhibition of MCT4 was sufficient to reduce lactate efflux in three-dimensional (3D) CRC spheroids but not in two-dimensional cell-cultures. Co-administration of the MCT4 inhibitor and ICB augmented immune cell infiltration, T-cell function and decreased CRC spheroid viability in a 3D co-culture model of human CRC spheroids with blood leukocytes. Accordingly, combination of MCT4 and ICB increased intratumoral pH, improved leukocyte infiltration and T-cell activation, delayed tumor growth, and prolonged survival in vivo. MCT1 inhibition exerted no further beneficial impact. Conclusions These findings demonstrate that single MCT4 inhibition represents a novel therapeutic approach to reverse lactic-acid driven immunosuppression and might be suitable to improve ICB efficacy

Natural Variation of Arabidopsis Root Architecture Reveals Complementing Adaptive Strategies to Potassium Starvation

Root architecture is a highly plastic and environmentally responsive trait that enables plants to counteract nutrient scarcities with different foraging strategies. In potassium (K) deficiency (low K), seedlings of the Arabidopsis (Arabidopsis thaliana) reference accession Columbia (Col-0) show a strong reduction of lateral root elongation. To date, it is not clear whether this is a direct consequence of the lack of K as an osmoticum or a triggered response to maintain the growth of other organs under limiting conditions. In this study, we made use of natural variation within Arabidopsis to look for novel root architectural responses to low K. A comprehensive set of 14 differentially responding root parameters were quantified in K-starved and K-replete plants. We identified a phenotypic gradient that links two extreme strategies of morphological adaptation to low K arising from a major tradeoff between main root (MR) and lateral root elongation. Accessions adopting strategy I (e. g. Col-0) maintained MR growth but compromised lateral root elongation, whereas strategy II genotypes (e.g. Catania-1) arrested MR elongation in favor of lateral branching. K resupply and histochemical staining resolved the temporal and spatial patterns of these responses. Quantitative trait locus analysis of K-dependent root architectures within a Col-0 3 Catania-1 recombinant inbred line population identified several loci each of which determined a particular subset of root architectural parameters. Our results indicate the existence of genomic hubs in the coordinated control of root growth in stress conditions and provide resources to facilitate the identification of the underlying genes