Search

Abstract Background Wild carnivores living alongside humans and domestic animals are vulnerable to changes in the infectious disease dynamics in their populations. The aims of this study were to determine the prevalence and diversity of selected tick-borne pathogens (TBPs) of veterinary and/or zoonotic concern in wild populations of caracals (Caracal caracal) occurring in human-modified landscapes in South Africa. Using molecular techniques, we screened 57 caracal blood samples for infection by rickettsial bacteria and piroplasms in three regions of South Africa: rangeland in the Central Karoo (n = 27) and Namaqualand (n = 14) as well as the urban edge of the Cape Peninsula (n = 16) of South Africa. To characterise pathogen identity, we sequenced the 18S rRNA and 16S rRNA genes from positive samples and analysed sequences within a phylogenetic framework. We also examine the diversity of potential tick vectors. Results All individuals tested were infected with at least one tick-borne pathogen. Pathogens included Hepatozoon felis, Babesia felis, Babesia leo and a potentially novel Babesia species. An Anaplasma species previously described in South African domestic dogs was also found in 88% of urban edge caracals. Higher rates of co-infection characterised urban edge caracals (81% vs 15% and 0% in the two rangeland populations), as well as a greater incidence of mixed infections. Host attached tick species include Haemaphysalis elliptica, an important pathogen vector among carnivore hosts. Conclusions This study confirms the occurrence of previously undocumented tick-borne pathogens infecting free-ranging caracals in human-modified landscapes. We identify clear differences in the pathogen profiles among our study populations and discuss the likely health costs to caracals living adjacent to urban areas.

In this article we determine the effect of an extralimital megaherbivore on the reproductive potential and vegetation structure of two keystone tree species in the Auob River in the south western Kalahari Desert of southern Africa. Using spoor and dung counts we establish the presence of giraffe in three predetermined density zones by walking 50 transects across the river in each zone. We also photographed six trees from each species in each zone and use these photographs to determine browse impact on reproductive potential, canopy volume as well as the percentage dieback on the extremities of the canopy. We then perform stable carbon and nitrogen isotope analysis on the leaves of the trees and compare these relative to the isotope ratios of giraffe dung to ascertain dietary preference. Crude protein was determined as a guide to nutritive value. Finally, we determine both chemical and physical defences for the two species. Our results show a significant negative impact of giraffe browse on tree canopies, no significant differences in recruitment and a noticeable decrease in flowers and pods at the giraffe browse height of 2 m – 5 m. No significant differences in crude protein or condensed tannins were found but significant differences in spinescence. Giraffe are not endemic to the Auob River and our study shows that the introduction of these animals is having a negative impact on the canopies of Vachellia haematoxylon. While there are, as yet, no significant impacts on reproductive potential we speculate that this will happen with time. Conservation implications: Our study shows that giraffe are significantly impacting the canopies of two common tree species in the Auob River in the arid Kgalagadi Transfrontier Park. Without management intervention an increasing population of giraffe will result in substantial changes to the plant community vegetation structure of the river.

Introduction: Although drought resistance of a plant may be achieved through morphological, structural, physiological, cellular, and molecular adaptations, most studies remain limited to quantifying the effect of drought on biomass., Methods: Using a highthroughput phenotypic imaging system, we evaluated the drought resistance of 151 bean accessions (Phaseolinae; Fabaceae) in an explorative approach, by quantifying five different traits simultaneously: biomass, water use efficiency (WUE), relative water content (RWC), chlorophyll content (NDVI), and root/shoot ratio. Since crop wild relatives are important resources for breeding programs, we analyzed both wild and cultivated accessions, most of which have never been evaluated for drought resistance before., Results: We demonstrate that the five traits are affected very differently by drought in the studied accessions, with significant correlations existing only between the biomass and WUE indicators (r=0.39), and between the RWC and NDVI indicators (r=0.40). When grouping accessions by subgenus or by species, large intraspecific and withinsubgenus variation was found. For this reason, we performed a cluster analysis, which grouped the accessions into five distinct clusters with similar response profiles. We also correlated the drought resistance for each accession to local climate variables at their original collection sites. The biomass, WUE, and RWC indicators were significantly correlated to annual precipitation (r=0.40, r=0.20, r=0.22, respectively), confirming that accessions from arid environments are generally more drought resistant., Discussion: Our results demonstrate that the drought resistance of Phaseolinae beans is a multifaceted characteristic and cannot be simply quantified through biomass. Furthermore, the broader knowledge of the drought resistance of the accessions studied here may prove an invaluable resource for future crop production., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Verheyen, Dhondt, Abbeloos, Eeckhout, Janssens, Leyns, Scheldeman, Storme and Vandelook.)

Although the plant kingdom provides an enormous diversity of metabolites with potentially beneficial applications for humankind, a large fraction of these metabolites and their biosynthetic pathways remain unknown. Resolving metabolite structures and their biosynthetic pathways is key to gaining biological understanding and to allow metabolic engineering. In order to retrieve novel biosynthetic genes involved in specialized metabolism, we developed a novel untargeted method designated as qualitative trait GWAS (QT-GWAS) that subjects qualitative metabolic traits to a genome-wide association study, while the conventional metabolite GWAS (mGWAS) mainly considers the quantitative variation of metabolites. As a proof of the validity of QT-GWAS, 23 and 15 of the retrieved associations identified in Arabidopsis thaliana by QT-GWAS and mGWAS, respectively, were supported by previous research. Furthermore, seven gene-metabolite associations retrieved by QT-GWAS were confirmed in this study through reverse genetics combined with metabolomics and/or in vitro enzyme assays. As such, we established that CYTOCHROME P450 706A5 (CYP706A5) is involved in the biosynthesis of chroman derivatives, UDP-GLYCOSYLTRANSFERASE 76C3 (UGT76C3) is able to hexosylate guanine in vitro and in planta, and SULFOTRANSFERASE 202B1 (SULT202B1) catalyzes the sulfation of neolignans in vitro. Collectively, our study demonstrates that the untargeted QT-GWAS method can retrieve valid gene-metabolite associations at the level of enzyme-encoding genes, even new associations that cannot be found by the conventional mGWAS, providing a new approach for dissecting qualitative metabolic traits., (Copyright © 2023 The Author. Published by Elsevier Inc. All rights reserved.)

The phenylpropanoid cinnamic acid (CA) is a plant metabolite that can occur under a trans - or cis -form. In contrast to the proven bioactivity of the cis -form ( c -CA), the activity of trans -CA ( t -CA) is still a matter of debate. We tested both compounds using a submerged rice coleoptile assay and demonstrated that they have opposite effects on cell elongation. Notably, in the tip of rice coleoptile t -CA showed an inhibiting and c -CA a stimulating activity. By combining transcriptomics and (untargeted) metabolomics with activity assays and genetic and pharmacological experiments, we aimed to explain the underlying mechanistic processes. We propose a model in which c -CA treatment activates proton pumps and stimulates acidification of the apoplast, which in turn leads to the loosening of the cell wall, necessary for elongation. We hypothesize that c -CA also inactivates auxin efflux transporters, which might cause a local auxin accumulation in the tip of the coleoptile. For t -CA, the phenotype can partially be explained by a stimulation of cell wall polysaccharide feruloylation, leading to a more rigid cell wall. Metabolite profiling also demonstrated that salicylic acid (SA) derivatives are increased upon t -CA treatment. As SA is a known antagonist of auxin, the shift in SA homeostasis provides an additional explanation of the observed t -CA-mediated restriction on cell growth., Competing Interests: The authors declare that there are no conflicts of interest., (© 2022 The Authors. Plant Direct published by American Society of Plant Biologists and the Society for Experimental Biology and John Wiley & Sons Ltd.)

Root system architecture (RSA) has a direct influence on the efficiency of nutrient uptake and plant growth, but the genetics of RSA are often studied only at the seedling stage. To get an insight into the genetic blueprint of a more mature RSA, we exploited natural variation and performed a detailed in vitro study of 241 Arabidopsis thaliana accessions using large petri dishes. A comprehensive analysis of 17 RSA traits showed high variability among the different accessions, unveiling correlations between traits and conditions of the natural habitat of the plants. A sub-selection of these accessions was grown in water-limiting conditions in a rhizotron set-up, which revealed that especially the spatial distribution showed a high consistency between in vitro and ex vitro conditions, while in particular, a large root area in the lower zone favored drought tolerance. The collected RSA phenotype data were used to perform genome-wide association studies (GWAS), which stands out from the previous studies by its exhaustive measurements of RSA traits on more mature Arabidopsis accessions used for GWAS. As a result, we found not only several genes involved in the lateral root (LR) development or auxin signaling pathways to be associated with RSA traits but also new candidate genes that are potentially involved in the adaptation to the natural habitats., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Deja-Muylle, Opdenacker, Parizot, Motte, Lobet, Storme, Clauw, Njo and Beeckman.)

The phenylpropanoid pathway serves a central role in plant metabolism, providing numerous compounds involved in diverse physiological processes. Most carbon entering the pathway is incorporated into lignin. Although several phenylpropanoid pathway mutants show seedling growth arrest, the role for lignin in seedling growth and development is unexplored. We use complementary pharmacological and genetic approaches to block CINNAMATE-4-HYDROXYLASE (C4H) functionality in Arabidopsis seedlings and a set of molecular and biochemical techniques to investigate the underlying phenotypes. Blocking C4H resulted in reduced lateral rooting and increased adventitious rooting apically in the hypocotyl. These phenotypes coincided with an inhibition in AUX transport. The upstream accumulation in cis-cinnamic acid was found to be likely to cause polar AUX transport inhibition. Conversely, a downstream depletion in lignin perturbed phloem-mediated AUX transport. Restoring lignin deposition effectively reestablished phloem transport and, accordingly, AUX homeostasis. Our results show that the accumulation of bioactive intermediates and depletion in lignin jointly cause the aberrant phenotypes upon blocking C4H, and demonstrate that proper deposition of lignin is essential for the establishment of AUX distribution in seedlings. Our data position the phenylpropanoid pathway and lignin in a new physiological framework, consolidating their importance in plant growth and development., (© 2021 The Authors New Phytologist © 2021 New Phytologist Foundation.)

Gene regulation is a dynamic process in which transcription factors (TFs) play an important role in controlling spatiotemporal gene expression. To enhance our global understanding of regulatory interactions in Arabidopsis thaliana, different regulatory input networks capturing complementary information about DNA motifs, open chromatin, TF-binding and expression-based regulatory interactions were combined using a supervised learning approach, resulting in an integrated gene regulatory network (iGRN) covering 1,491 TFs and 31,393 target genes (1.7 million interactions). This iGRN outperforms the different input networks to predict known regulatory interactions and has a similar performance to recover functional interactions compared to state-of-the-art experimental methods. The iGRN correctly inferred known functions for 681 TFs and predicted new gene functions for hundreds of unknown TFs. For regulators predicted to be involved in reactive oxygen species (ROS) stress regulation, we confirmed in total 75% of TFs with a function in ROS and/or physiological stress responses. This includes 13 ROS regulators, previously not connected to any ROS or stress function, that were experimentally validated in our ROS-specific phenotypic assays of loss- or gain-of-function lines. In conclusion, the presented iGRN offers a high-quality starting point to enhance our understanding of gene regulation in plants by integrating different experimental data types.

Plant cells perceive and adapt to an ever-changing environment by modifying their plasma membrane (PM) proteome. Whereas secretion deposits new integral membrane proteins, internalization by endocytosis removes membrane proteins and associated ligands, largely with the aid of adaptor protein (AP) complexes and the scaffolding molecule clathrin. Two AP complexes function in clathrin-mediated endocytosis at the PM in plant cells, the heterotetrameric AP-2 complex and the hetero-octameric TPLATE complex (TPC). Whereas single subunit mutants in AP-2 develop into viable plants, genetic mutation of a single TPC subunit causes fully penetrant male sterility and silencing single subunits leads to seedling lethality. To address TPC function in somatic root cells, while minimizing indirect effects on plant growth, we employed nanobody-dependent delocalization of a functional, GFP-tagged TPC subunit, TML, in its respective homozygous genetic mutant background. In order to decrease the amount of functional TPC at the PM, we targeted our nanobody construct to the mitochondria and fused it to TagBFP2 to visualize it independently of its bait. We furthermore limited the effect of our delocalization to those tissues that are easily accessible for live-cell imaging by expressing it from the PIN2 promoter, which is active in root epidermal and cortex cells. With this approach, we successfully delocalized TML from the PM. Moreover, we also show co-recruitment of TML-GFP and AP2A1-TagRFP to the mitochondria, suggesting that our approach delocalized complexes, rather than individual adaptor complex subunits. In line with the specific expression domain, we only observed minor effects on root growth, yet realized a clear reduction of endocytic flux in epidermal root cells. Nanobody-dependent delocalization in plants, here exemplified using a TPC subunit, has the potential to be widely applicable to achieve specific loss-of-function analysis of otherwise lethal mutants., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Winkler, De Meyer, Mylle, Storme, Grones and Van Damme.)

Leaf growth is a complex trait for which many similarities exist in different plant species, suggesting functional conservation of the underlying pathways. However, a global view of orthologous genes involved in leaf growth showing conserved expression in dicots and monocots is currently missing. Here, we present a genome-wide comparative transcriptome analysis between Arabidopsis and maize, identifying conserved biological processes and gene functions active during leaf growth. Despite the orthology complexity between these distantly related plants, 926 orthologous gene groups including 2829 Arabidopsis and 2974 maize genes with similar expression during leaf growth were found, indicating conservation of the underlying molecular networks. We found 65% of these genes to be involved in one-to-one orthology, whereas only 28.7% of the groups with divergent expression had one-to-one orthology. Within the pool of genes with conserved expression, 19 transcription factor families were identified, demonstrating expression conservation of regulators active during leaf growth. Additionally, 25 Arabidopsis and 25 maize putative targets of the TCP transcription factors with conserved expression were determined based on the presence of enriched transcription factor binding sites. Based on large-scale phenotypic data, we observed that genes with conserved expression have a higher probability to be involved in leaf growth and that leaf-related phenotypes are more frequently present for genes having orthologues between dicots and monocots than clade-specific genes. This study shows the power of integrating transcriptomic with orthology data to identify or select candidates for functional studies during leaf development in flowering plants., (© 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

The target of rapamycin (TOR) kinase is a conserved regulatory hub that translates environmental and nutritional information into permissive or restrictive growth decisions. Despite the increased appreciation of the essential role of the TOR complex in plants, no large-scale phosphoproteomics or interactomics studies have been performed to map TOR signalling events in plants. To fill this gap, we combined a systematic phosphoproteomics screen with a targeted protein complex analysis in the model plant Arabidopsis thaliana. Integration of the phosphoproteome and protein complex data on the one hand shows that both methods reveal complementary subspaces of the plant TOR signalling network, enabling proteome-wide discovery of both upstream and downstream network components. On the other hand, the overlap between both data sets reveals a set of candidate direct TOR substrates. The integrated network embeds both evolutionarily-conserved and plant-specific TOR signalling components, uncovering an intriguing complex interplay with protein synthesis. Overall, the network provides a rich data set to start addressing fundamental questions about how TOR controls key processes in plants, such as autophagy, auxin signalling, chloroplast development, lipid metabolism, nucleotide biosynthesis, protein translation or senescence.

The conserved poly(ADP-ribosyl)ation (PAR) pathway consists of three genetic components that are potential targets to modulate the plant's energy homeostasis upon stress with the aim to improve yield stability in crops and help secure food supply. We studied the role of the PAR pathway component ADP-ribose/NADH pyrophosphohydrolase (AtNUDX7) in yield and mild drought stress by using a transgenic approach in Arabidopsis thaliana and maize (Zea mays). Arabidopsis AtNUDX7 cDNA was overexpressed in Arabidopsis and maize by means of the constitutive Cauliflower Mosaic Virus 35S promoter and the strong constitutive Brachypodium distachyon pBdEF1α promoter, respectively. Overexpression of AtNUDX7 in Arabidopsis improved seed parameters that were measured by a novel, automated method, accelerated flowering and reduced inflorescence height. This combination of beneficial traits suggested that AtNUDX7 overexpression in Arabidopsis might enhance the ADP-ribose recycling step and maintain energy levels by supplying an ATP source in the poly(ADP-ribosyl)ation energy homeostasis pathway. Arabidopsis and maize lines with high, medium and low overexpression levels of the AtNUDX7 gene were analysed in automated platforms and the inhibition of several growth parameters was determined under mild drought stress conditions. The data showed that the constitutive overexpression of the Arabidopsis AtNUDX7 gene in Arabidopsis and maize at varying levels did not improve tolerance to mild drought stress, but knocking down AtNUDX7 expression did, however at the expense of general growth under normal conditions.

Yeast two-hybrid (Y2H) is a well-established genetics-based system that uses yeast to selectively display binary protein-protein interactions (PPIs). To meet the current need to unravel complex PPI networks, several adaptations have been made to establish medium- to high-throughput Y2H screening platforms, with several having successfully incorporated the use of the next-generation sequencing (NGS) technology to increase the scale and sensitivity of the method. However, these have been to date mainly restricted to the use of fully annotated custom-made open reading frame (ORF) libraries and subject to complex downstream data processing. Here, a streamlined Y2H library screening strategy, based on integration of Y2H with NGS, called Y2H-seq, was developed, which allows efficient and reliable screening of Y2H cDNA libraries. To generate proof of concept, the method was applied to screen for interaction partners of two key components of the jasmonate signaling machinery in the model plant Arabidopsis thaliana, resulting in the identification of several previously reported as well as hitherto unknown interactors. Our Y2H-seq method offers a user-friendly, specific and sensitive screening method that allows identification of PPIs without prior knowledge of the organism's ORFs, thereby extending the method to organisms of which the genome has not entirely been annotated yet. The quantitative NGS readout allows to increase genome coverage, thereby overcoming some of the bottlenecks of current Y2H technologies, which will further strengthen the value of the Y2H technology as a discovery platform., Competing Interests: The authors have declared that no competing interests exist.

Somatic polyploidy caused by endoreplication is observed in arthropods, molluscs, and vertebrates but is especially prominent in higher plants, where it has been postulated to be essential for cell growth and fate maintenance. However, a comprehensive understanding of the physiological significance of plant endopolyploidy has remained elusive. Here, we modeled and experimentally verified a high-resolution DNA endoploidy map of the developing Arabidopsis thaliana root, revealing a remarkable spatiotemporal control of DNA endoploidy levels across tissues. Fitting of a simplified model to publicly available data sets profiling root gene expression under various environmental stress conditions suggested that this root endoploidy patterning may be stress-responsive. Furthermore, cellular and transcriptomic analyses revealed that inhibition of endoreplication onset alters the nuclear-to-cellular volume ratio and the expression of cell wall-modifying genes, in correlation with the appearance of cell structural changes. Our data indicate that endopolyploidy might serve to coordinate cell expansion with structural stability and that spatiotemporal endoreplication pattern changes may buffer for stress conditions, which may explain the widespread occurrence of the endocycle in plant species growing in extreme or variable environments., (© 2018 American Society of Plant Biologists. All rights reserved.)

High-throughput RNA sequencing has proven invaluable not only to explore gene expression but also for both gene prediction and genome annotation. However, RNA sequencing, carried out on tens or even hundreds of samples, requires easy and cost-effective sample preparation methods using minute RNA amounts. Here, we present TranSeq, a high-throughput 3'-end sequencing procedure that requires 10- to 20-fold fewer sequence reads than the current transcriptomics procedures. TranSeq significantly reduces costs and allows a greater increase in size of sample sets analyzed in a single experiment. Moreover, in comparison with other 3'-end sequencing methods reported to date, we demonstrate here the reliability and immediate applicability of TranSeq and show that it not only provides accurate transcriptome profiles but also produces precise expression measurements of specific gene family members possessing high sequence similarity. This is difficult to achieve in standard RNA-seq methods, in which sequence reads cover the entire transcript. Furthermore, mapping TranSeq reads to the reference tomato genome facilitated the annotation of new transcripts improving >45% of the existing gene models. Hence, we anticipate that using TranSeq will boost large-scale transcriptome assays and increase the spatial and temporal resolution of gene expression data, in both model and non-model plant species. Moreover, as already performed for tomato (ITAG3.0; www.solgenomics.net), we strongly advocate its integration into current and future genome annotations., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Small GTP-binding proteins from the ADP-ribosylation factor (ARF) family are important regulators of vesicle formation and cellular trafficking in all eukaryotes. ARF activation is accomplished by a protein family of guanine nucleotide exchange factors (GEFs) that contain a conserved catalytic Sec7 domain. Here, we identified and characterized Secdin, a small-molecule inhibitor of Arabidopsis thaliana ARF-GEFs. Secdin application caused aberrant retention of plasma membrane (PM) proteins in late endosomal compartments, enhanced vacuolar degradation, impaired protein recycling, and delayed secretion and endocytosis. Combined treatments with Secdin and the known ARF-GEF inhibitor Brefeldin A (BFA) prevented the BFA-induced PM stabilization of the ARF-GEF GNOM, impaired its translocation from the Golgi to the trans -Golgi network/early endosomes, and led to the formation of hybrid endomembrane compartments reminiscent of those in ARF-GEF-deficient mutants. Drug affinity-responsive target stability assays revealed that Secdin, unlike BFA, targeted all examined Arabidopsis ARF-GEFs, but that the interaction was probably not mediated by the Sec7 domain because Secdin did not interfere with the Sec7 domain-mediated ARF activation. These results show that Secdin and BFA affect their protein targets through distinct mechanisms, in turn showing the usefulness of Secdin in studies in which ARF-GEF-dependent endomembrane transport cannot be manipulated with BFA., (© 2018 American Society of Plant Biologists. All rights reserved.)