A genome-wide association study reveals cytokinin as a major component in the root defense responses against Ralstonia solanacearum.

Bacterial wilt caused by the soil-borne pathogen Ralstonia solancearum is economically devastating, with no effective methods to fight the disease. This pathogen invades plants through their roots and colonizes their xylem, clogging the vasculature and causing rapid wilting. Key to preventing colonization are the early defense responses triggered in the host's root upon infection, which remain mostly unknown. Here, we have taken advantage of a high-throughput in vitro infection system to screen natural variability associated with the root growth inhibition phenotype caused by R. solanacearum in Arabidopsis during the first hours of infection. To analyze the genetic determinants of this trait, we have performed a genome-wide association study, identifying allelic variation at several loci related to cytokinin metabolism, including genes responsible for biosynthesis and degradation of cytokinin. Further, our data clearly demonstrate that cytokinin signaling is induced early during the infection process and cytokinin contributes to immunity against R. solanacearum. This study highlights a new role for cytokinin in root immunity, paving the way for future research that will help in understanding the mechanisms underpinning root defenses.

Ribosome assembly factor Adenylate Kinase 6 maintains cell proliferation and cell size homeostasis during root growth.

From the cellular perspective, organ growth is determined by production and growth of cells. Uncovering how these two processes are coordinated is essential for understanding organogenesis and regulation of organ growth. We utilized phenotypic and genetic variation of 252 natural accessions of Arabidopsis thaliana to conduct genome-wide association studies (GWAS) for identifying genes underlying root growth variation; using a T-DNA line candidate approach, we identified one gene involved in root growth control and characterized its function using microscopy, root growth kinematics, G2/M phase cell count, ploidy levels and ribosome polysome profiles. We identified a factor contributing to root growth control: Arabidopsis Adenylate Kinase 6 (AAK6). AAK6 is required for normal cell production and normal cell elongation, and its natural genetic variation is involved in determining root growth differences between Arabidopsis accessions. A lack of AAK6 reduces cell production in the aak6 root apex, but this is partially compensated for by longer mature root cells. Thereby, aak6 mutants exhibit compensatory cell enlargement, a phenomenon unexpected in roots. Moreover, aak6 plants accumulate 80S ribosomes while the polysome profile remains unchanged, consistent with a phenotype of perturbed ribosome biogenesis. In conclusion, AAK6 impacts ribosome abundance, cell production and thereby root growth.

Identification of novel genes involved in phosphate accumulation in Lotus japonicus through Genome Wide Association mapping of root system architecture and anion content.

Phosphate represents a major limiting factor for plant productivity. Plants have evolved different solutions to adapt to phosphate limitation ranging from a profound tuning of their root system architecture and metabolic profile to the evolution of widespread mutualistic interactions. Here we elucidated plant responses and their genetic basis to different phosphate levels in a plant species that is widely used as a model for AM symbiosis: Lotus japonicus. Rather than focussing on a single model strain, we measured root growth and anion content in response to different levels of phosphate in 130 Lotus natural accessions. This allowed us not only to uncover common as well as divergent responses within this species, but also enabled Genome Wide Association Studies by which we identified new genes regulating phosphate homeostasis in Lotus. Among them, we showed that insertional mutants of a cytochrome B5 reductase and a Leucine-Rich-Repeat receptor showed different phosphate concentration in plants grown under phosphate sufficient condition. Under low phosphate conditions, we found a correlation between plant biomass and the decrease of plant phosphate concentration in plant tissues, representing a dilution effect. Altogether our data of the genetic and phenotypic variation within a species capable of AM complements studies that have been conducted in Arabidopsis, and advances our understanding of the continuum of genotype by phosphate level interaction existing throughout dicot plants.

GSNOR provides plant tolerance to iron toxicity via preventing iron-dependent nitrosative and oxidative cytotoxicity.

Iron (Fe) is essential for life, but in excess can cause oxidative cytotoxicity through the generation of Fe-catalyzed reactive oxygen species. It is yet unknown which genes and mechanisms can provide Fe-toxicity tolerance. Here, we identify S-nitrosoglutathione-reductase (GSNOR) variants underlying a major quantitative locus for root tolerance to Fe-toxicity in Arabidopsis using genome-wide association studies and allelic complementation. These variants act largely through transcript level regulation. We further show that the elevated nitric oxide is essential for Fe-dependent redox toxicity. GSNOR maintains root meristem activity and prevents cell death via inhibiting Fe-dependent nitrosative and oxidative cytotoxicity. GSNOR is also required for root tolerance to Fe-toxicity throughout higher plants such as legumes and monocots, which exposes an opportunity to address crop production under high-Fe conditions using natural GSNOR variants. Overall, this study shows that genetic or chemical modulation of the nitric oxide pathway can broadly modify Fe-toxicity tolerance.

Automated High-Throughput Root Phenotyping of Arabidopsis thaliana Under Nutrient Deficiency Conditions.

The central question of genetics is how a genotype determines the phenotype of an organism. Genetic mapping approaches are a key for finding answers to this question. In particular, genome-wide association (GWA) studies have been rapidly adopted to study the architecture of complex quantitative traits. This was only possible due to the improvement of high-throughput and low-cost phenotyping methodologies. In this chapter we provide a detailed protocol for obtaining root trait data from the model species Arabidopsis thaliana using the semiautomated, high-throughput phenotyping pipeline BRAT (Busch-lab Root Analysis Toolchain) for early root growth under the stress condition of iron deficiency. Extracted root trait data can be directly used to perform GWA mapping using the freely accessible web application GWAPP to identify marker polymorphisms associated with the phenotype of interest.

Large-Scale Phenotyping of Root Traits in the Model Legume Lotus japonicus.

Plants are sessile organisms that can tune their body architecture to the environment. This is very pronounced in their root system. In particular, nutrient availability strongly influences the architecture of the root system; depending on the abundance of specific nutrients, root growth rates and lateral root number are modulated. The extent of these effects is important for plant adaptation and has a major impact on plant fitness. However, the assessment of quantitative effects on a scale large enough for identifying genes and variants using quantitative genetics is difficult, and well-developed methods have been largely restricted to the model species Arabidopsis thaliana. In this chapter, we present a protocol for high-throughput phenotyping of early root traits in the model legume plant Lotus japonicus. This species allows for the study of important root-associated traits that are not present in Arabidopsis, such as symbioses with nitrogen-fixing Rhizobia and arbuscular mycorrhizal fungi. The methods described in this chapter can be used in the context of reverse and forward genetics approaches to dissect the genetic basis of root growth in legumes.

Long-Term Confocal Imaging of Arabidopsis thaliana Roots for Simultaneous Quantification of Root Growth and Fluorescent Signals.

Observing cellular and molecular processes in living organisms is key for understanding many important biological processes. Confocal microscopy is excellently suited for this as it enables the observation of molecules and cells in tissue layers of living organisms in three dimensions over time. However, in continuously growing organs, such as plant roots, observations over extended time spans become difficult as the specimen quickly grows out the field of view. Here, we provide a protocol that allows for the acquisition of confocal microscope time-lapse images of root tips spanning many hours, as the growing root tip is tracked and the microscopy is automatized to change the position of the stage. Importantly, due to its specific setup, this protocol allows for observing the effects of chemical stimuli or for creating specific growth conditions by precisely defining the growth medium during imaging. The protocol is suitable for observing multiple fluorophores, thereby moving beyond the level of individual genes. It is also simple enough to conduct larger numbers of these assays. Here we exemplify our method by describing the observation of root growth and GFP intensity in root tips under iron depletion conditions.

Genome-wide association mapping in plants exemplified for root growth in Arabidopsis thaliana.

Genome-wide association (GWA) mapping is a powerful technique to address the molecular basis of genotype to phenotype relationships and to map regulators of biological processes. This chapter presents a protocol for genome-wide association mapping in Arabidopsis thaliana using the user-friendly internet application GWAPP, and provides a specific protocol for acquiring root trait data suitable for GWA studies using the semi-automated, high-throughput phenotyping pipeline BRAT for early root growth.

A Scalable Open-Source Pipeline for Large-Scale Root Phenotyping of Arabidopsis.

Large-scale phenotyping of multicellular organisms is one of the current challenges in biology. We present a comprehensive and scalable pipeline that allows for the efficient phenotyping of root growth traits on a large scale. This includes a high-resolution, low-cost acquisition setup as well as the automated image processing software BRAT. We assess the performance of this pipeline in Arabidopsis thaliana under multiple growth conditions and show its utility by performing genome-wide association studies on 16 root growth traits quantified by BRAT each day during a 5-d time-course experiment. The most significantly associated genome region for root growth rate is a locus encoding a calcium sensing receptor. We find that loss of function and overexpression of this gene can significantly alter root growth in a growth condition dependent manner and that the minor natural allele of the Calcium Sensor Receptor locus is highly significantly enriched in populations in coastal areas, demonstrating the power of our approach to identify regulators of root growth that might have adaptive relevance.