A High-Throughput Approach for Photosynthesis Studies in a Brassicaceae Panel.

The study of natural variations in photosynthesis in the Brassicaceae family offers the possibility of identifying mechanisms to enhance photosynthetic efficiency in crop plants. Indeed, this family, and particularly its tribe Brassiceae, has been shown to harbor species that have a higher-than-expected photosynthetic efficiency, possibly as a result of a complex evolutionary history. Over the past two decades, methods have been developed to measure photosynthetic efficiency based on chlorophyll fluorescence. Chlorophyll fluorescence measurements are performed with special cameras, such as the FluorCams, which can be included in robotic systems to create high-throughput phenotyping platforms. While these platforms have so far demonstrated high efficiency in measuring small model species like Arabidopsis thaliana, they have the drawback of limited adaptability to accommodate different plant sizes. As a result, the range of species that can be analyzed is restricted. This chapter presents our approach to analyze the photosynthetic parameters: ϕPSII and Fv/Fm for a panel of Brassicaceae species, including a high-photosynthesis species, Hirschfeldia incana, and the adaptations to the phenotyping platform that are required to accommodate this varied group of plants.

Unveiling Nanoscale Heterogeneities at the Bias-Dependent Gold-Electrolyte Interface.

Electrified solid-liquid interfaces (SLIs) are extremely complex and dynamic, affecting both the dynamics and selectivity of reaction pathways at electrochemical interfaces. Enabling access to the structure and arrangement of interfacial water in situ with nanoscale resolution is essential to develop efficient electrocatalysts. Here, we probe the SLI energy of a polycrystalline Au(111) electrode in a neutral aqueous electrolyte through in situ electrochemical atomic force microscopy. We acquire potential-dependent maps of the local interfacial adhesion forces, which we associate with the formation energy of the electric double layer. We observe nanoscale inhomogeneities of interfacial adhesion force across the entire map area, indicating local differences in the ordering of the solvent/ions at the interface. Anion adsorption has a clear influence on the observed interfacial adhesion forces. Strikingly, the adhesion forces exhibit potential-dependent hysteresis, which depends on the local gold grain curvature. Our findings on a model electrode extend the use of scanning probe microscopy to gain insights into the local molecular arrangement of the SLI in situ, which can be extended to other electrocatalysts.

Genome-wide association analysis reveals genes controlling an antagonistic effect of biotic and osmotic stress on Arabidopsis thaliana growth.

While the response of Arabidopsis thaliana to drought, herbivory or fungal infection has been well-examined, the consequences of exposure to a series of such (a)biotic stresses are not well studied. This work reports on the genetic mechanisms underlying the Arabidopsis response to single osmotic stress, and to combinatorial stress, either fungal infection using Botrytis cinerea or herbivory using Pieris rapae caterpillars followed by an osmotic stress treatment. Several small-effect genetic loci associated with rosette dry weight (DW), rosette water content (WC), and the projected rosette leaf area in response to combinatorial stress were mapped using univariate and multi-environment genome-wide association approaches. A single-nucleotide polymorphism (SNP) associated with DROUGHT-INDUCED 19 (DI19) was identified by both approaches, supporting its potential involvement in the response to combinatorial stress. Several SNPs were found to be in linkage disequilibrium with known stress-responsive genes such as PEROXIDASE 34 (PRX34), BASIC LEUCINE ZIPPER 25 (bZIP25), RESISTANCE METHYLATED GENE 1 (RMG1) and WHITE RUST RESISTANCE 4 (WRR4). An antagonistic effect between biotic and osmotic stress was found for prx34 and arf4 mutants, which suggests PRX34 and ARF4 play an important role in the response to the combinatorial stress.

House dust mite sublingual allergen immunotherapy tablet is safe and well-tolerated in Dutch clinical practice.

Background: Half (49%) of clinically diagnosed allergic rhinitis (AR) patients are sensitized to house dust mite (HDM). If allergen avoidance and symptomatic medication fail, allergen immunotherapy may be indicated. Objective: We investigated safety and tolerability of HDM-sublingual immunotherapy by HDM-SLIT tablets in Dutch daily clinical practice. Methods: Daily intake of 12 SQ-HDM SLIT-tablet was investigated in a prospective, multicenter, observational study (EUPAS43753). It comprised 4 consultations in 1 year. Data on safety, tolerability, treatment satisfaction, symptomatic medication, compliance, and clinical effectiveness (Control of Allergic Rhinitis and Asthma Test; CARAT) were collected. Descriptive and longitudinal regression data analysis were performed. Results: Adult patients (n = 415), mean (SD) age 36.6 (12.2) years, 61.4% female and 36% asthmatic were included. The preponderance (65.1%) experienced adverse events (AEs). These, mostly mild (67%), AEs comprised: oral allergic reactions (58.6%), respiratory (12.4%) and gastrointestinal symptoms (9.4%). Sixty (14.5%) patients stopped due to AEs and 76 (18.3%) for non-AE reasons. CARAT scores improved clinically significant by 6 points and symptomatic medication use decreased from 96.1% to 77.4%. Most patients (74.5%) tolerated the treatment and were compliant (>86.5%). The majority of patients (62.4%) and investigators (69.4%) were satisfied with treatment. Conclusions: HDM SLIT-tablet is a safe and well-tolerated AR treatment. AEs occur often but are mostly mild and decreasing during the first year. CARAT scores improved and symptomatic medication use decreased suggesting better control of AR with treatment. Compliance, tolerability, and treatment satisfaction are good. However, patient follow-up and compliance remain important points of attention when initiating treatment.

LeTra: a leaf tracking workflow based on convolutional neural networks and intersection over union.

BACKGROUND: The study of plant photosynthesis is essential for productivity and yield. Thanks to the development of high-throughput phenotyping (HTP) facilities, based on chlorophyll fluorescence imaging, photosynthetic traits can be measured in a reliable, reproducible and efficient manner. In most state-of-the-art HTP platforms, these traits are automatedly analyzed at individual plant level, but information at leaf level is often restricted by the use of manual annotation. Automated leaf tracking over time is therefore highly desired. Methods for tracking individual leaves are still uncommon, convoluted, or require large datasets. Hence, applications and libraries with different techniques are required. New phenotyping platforms are initiated now more frequently than ever; however, the application of advanced computer vision techniques, such as convolutional neural networks, is still growing at a slow pace. Here, we provide a method for leaf segmentation and tracking through the fine-tuning of Mask R-CNN and intersection over union as a solution for leaf tracking on top-down images of plants. We also provide datasets and code for training and testing on both detection and tracking of individual leaves, aiming to stimulate the community to expand the current methodologies on this topic. RESULTS: We tested the results for detection and segmentation on 523 Arabidopsis thaliana leaves at three different stages of development from which we obtained a mean F-score of 0.956 on detection and 0.844 on segmentation overlap through the intersection over union (IoU). On the tracking side, we tested nine different plants with 191 leaves. A total of 161 leaves were tracked without issues, accounting to a total of 84.29% correct tracking, and a Higher Order Tracking Accuracy (HOTA) of 0.846. In our case study, leaf age and leaf order influenced photosynthetic capacity and photosynthetic response to light treatments. Leaf-dependent photosynthesis varies according to the genetic background. CONCLUSION: The method provided is robust for leaf tracking on top-down images. Although one of the strong components of the method is the low requirement in training data to achieve a good base result (based on fine-tuning), most of the tracking issues found could be solved by expanding the training dataset for the Mask R-CNN model.

NICOTIANAMINE SYNTHASE activity affects nucleolar iron accumulation and impacts rDNA silencing and RNA methylation in Arabidopsis.

In plant cells, a large pool of iron (Fe) is contained in the nucleolus, as well as in chloroplasts and mitochondria. A central determinant for intracellular distribution of Fe is nicotianamine (NA) generated by NICOTIANAMINE SYNTHASE (NAS). Here, we used Arabidopsis thaliana plants with disrupted NAS genes to study the accumulation of nucleolar iron and understand its role in nucleolar functions and more specifically in rRNA gene expression. We found that nas124 triple mutant plants, which contained lower quantities of the iron ligand NA, also contained less iron in the nucleolus. This was concurrent with the expression of normally silenced rRNA genes from nucleolar organizer regions 2 (NOR2). Notably, in nas234 triple mutant plants, which also contained lower quantities of NA, nucleolar iron and rDNA expression were not affected. In contrast, in both nas124 and nas234, specific RNA modifications were differentially regulated in a genotype dependent manner. Taken together, our results highlight the impact of specific NAS activities in RNA gene expression. We discuss the interplay between NA and nucleolar iron with rDNA functional organization and RNA methylation.

Optical Characterization of Plasmonic Indium Lattices Fabricated via Electrochemical Deposition.

The optical properties of periodic metallic nanoparticle lattices have found many exciting applications. Indium is an emerging plasmonic material that offers to extend the plasmonic applications given by gold and silver from the visible to the ultraviolet spectral range, with applications in imaging, sensing, and lasing. Due to the high vapor pressure/low melting temperature of indium, nanofabrication of ordered metallic nanoparticles is nontrivial. In this work, we show the potential of selective area electrochemical deposition to generate large-area lattices of In pillars for plasmonic applications. We study the optical response of the In lattices by means of angle-dependent extinction measurements demonstrating strong plasmonic surface lattice resonances and a good agreement with numerical simulations. The results open avenues toward high-quality lattices of plasmonic indium nanoparticles and can be extended to other promising plasmonic materials that can be electrochemically grown.

A genome-wide association study identifies novel players in Na and Fe homeostasis in Arabidopsis thaliana under alkaline-salinity stress.

In nature, multiple stress factors occur simultaneously. The screening of natural diversity panels and subsequent Genome-Wide Association Studies (GWAS) is a powerful approach to identify genetic components of various stress responses. Here, the nutritional status variation of a set of 270 natural accessions of Arabidopsis thaliana grown on a natural saline-carbonated soil is evaluated. We report significant natural variation on leaf Na (LNa) and Fe (LFe) concentrations in the studied accessions. Allelic variation in the NINJA and YUC8 genes is associated with LNa diversity, and variation in the ALA3 is associated with LFe diversity. The allelic variation detected in these three genes leads to changes in their mRNA expression and correlates with plant differential growth performance when plants are exposed to alkaline salinity treatment under hydroponic conditions. We propose that YUC8 and NINJA expression patters regulate auxin and jasmonic signaling pathways affecting plant tolerance to alkaline salinity. Finally, we describe an impairment in growth and leaf Fe acquisition associated with differences in root expression of ALA3, encoding a phospholipid translocase active in plasma membrane and the trans Golgi network which directly interacts with proteins essential for the trafficking of PIN auxin transporters, reinforcing the role of phytohormonal processes in regulating ion homeostasis under alkaline salinity.

Ion Current Rectification and Long-Range Interference in Conical Silicon Micropores.

Fluidic devices exhibiting ion current rectification (ICR), or ionic diodes, are of broad interest for applications including desalination, energy harvesting, and sensing, among others. For such applications a large conductance is desirable, which can be achieved by simultaneously using thin membranes and wide pores. In this paper we demonstrate ICR in micrometer sized conical channels in a thin silicon membrane with pore diameters comparable to the membrane thickness but both much larger than the electrolyte screening length. We show that for these pores the entrance resistance is key not only to Ohmic conductance around 0 V but also for understanding ICR, both of which we measure experimentally and capture within a single analytic theoretical framework. The only fit parameter in this theory is the membrane surface potential, for which we find that it is voltage dependent and its value is excessively large compared to the literature. From this we infer that surface charge outside the pore strongly contributes to the observed Ohmic conductance and rectification by a different extent. We experimentally verify this hypothesis in a small array of pores and find that ICR vanishes due to pore-pore interactions mediated through the membrane surface, while Ohmic conductance around 0 V remains unaffected. We find that the pore-pore interaction for ICR is set by a long-ranged decay of the concentration which explains the surprising finding that the ICR vanishes for even a sparsely populated array with a pore-pore spacing as large as 7 µm.

Micro-analytical and molecular approaches for understanding the distribution, biochemistry, and molecular biology of selenium in (hyperaccumulator) plants.

MAIN CONCLUSION: Micro-analytical techniques to untangle Se distribution and chemical speciation in plants coupled with molecular biology analysis enable the deciphering of metabolic pathways responsible for Se tolerance and accumulation. Selenium (Se) is not essential for plants and is toxic at high concentrations. However, Se hyperaccumulator plants have evolved strategies to both tolerate and accumulate > 1000 µg Se g-1 DW in their living above-ground tissues. Given the complexity of the biochemistry of Se, various approaches have been adopted to study Se metabolism in plants. These include X-ray-based techniques for assessing distribution and chemical speciation of Se, and molecular biology techniques to identify genes implicated in Se uptake, transport, and assimilation. This review presents these techniques, synthesises the current state of knowledge on Se metabolism in plants, and highlights future directions for research into Se (hyper)accumulation and tolerance. We conclude that powerful insights may be gained from coupling information on the distribution and chemical speciation of Se to genome-scale studies to identify gene functions and molecular mechanisms that underpin Se tolerance and accumulation in these ecologically and biotechnologically important plants species. The study of Se metabolism is challenging and is a useful testbed for developing novel analytical approaches that are potentially more widely applicable to the study of the regulation of a wide range of metal(loid)s in hyperaccumulator plants.

The genome sequence of Hirschfeldia incana, a new Brassicaceae model to improve photosynthetic light-use efficiency.

Photosynthesis is a key process in sustaining plant and human life. Improving the photosynthetic capacity of agricultural crops is an attractive means to increase their yields. While the core mechanisms of photosynthesis are highly conserved in C3 plants, these mechanisms are very flexible, allowing considerable diversity in photosynthetic properties. Among this diversity is the maintenance of high photosynthetic light-use efficiency at high irradiance as identified in a small number of exceptional C3 species. Hirschfeldia incana, a member of the Brassicaceae family, is such an exceptional species, and because it is easy to grow, it is an excellent model for studying the genetic and physiological basis of this trait. Here, we present a reference genome of H. incana and confirm its high photosynthetic light-use efficiency. While H. incana has the highest photosynthetic rates found so far in the Brassicaceae, the light-saturated assimilation rates of closely related Brassica rapa and Brassica nigra are also high. The H. incana genome has extensively diversified from that of B. rapa and B. nigra through large chromosomal rearrangements, species-specific transposon activity, and differential retention of duplicated genes. Duplicated genes in H. incana, B. rapa, and B. nigra that are involved in photosynthesis and/or photoprotection show a positive correlation between copy number and gene expression, providing leads into the mechanisms underlying the high photosynthetic efficiency of these species. Our work demonstrates that the H. incana genome serves as a valuable resource for studying the evolution of high photosynthetic light-use efficiency and enhancing photosynthetic rates in crop species.

Construction and analysis of a Noccaea caerulescens TILLING population.

BACKGROUND: Metals such as Zn or Cd are toxic to plant and humans when they are exposed in high quantities through contaminated soil or food. Noccaea caerulescens, an extraordinary Zn/Cd/Ni hyperaccumulating species, is used as a model plant for metal hyperaccumulation and phytoremediation studies. Current reverse genetic techniques to generate mutants based on transgenesis is cumbersome due to the low transformation efficiency of this species. We aimed to establish a mutant library for functional genomics by a non-transgenic approach, to identify mutants with an altered mineral profiling, and to screen for mutations in bZIP19, a regulator of Zn homeostasis in N. caerulescens. RESULTS: To generate the N. caerulescens mutant library, 3000 and 5000 seeds from two sister plants of a single-seed recurrent inbred descendant of the southern French accession Saint-Félix-de-Pallières (SF) were mutagenized respectively by 0.3 or 0.4% ethyl methane sulfonate (EMS). Two subpopulations of 5000 and 7000 M2 plants were obtained after 0.3 or 0.4% EMS treatment. The 0.4% EMS treatment population had a higher mutant frequency and was used for TILLING. A High Resolution Melting curve analysis (HRM) mutation screening platform was optimized and successfully applied to detect mutations for NcbZIP19, encoding a transcription factor controlling Zn homeostasis. Of four identified point mutations in NcbZIP19, two caused non-synonymous substitutions, however, these two mutations did not alter the ionome profile compared to the wild type. Forward screening of the 0.4% EMS treatment population by mineral concentration analysis (ionomics) in leaf material of each M2 plant revealed putative mutants affected in the concentration of one or more of the 20 trace elements tested. Several of the low-Zn mutants identified in the ionomic screen did not give progeny, illustrating the importance of Zn for the species. The mutant frequency of the population was evaluated based on an average of 2.3 knockout mutants per tested monogenic locus. CONCLUSIONS: The 0.4% EMS treatment population is effectively mutagenized suitable for forward mutant screens and TILLING. Difficulties in seed production in low Zn mutants, obtained by both forward and reverse genetic approach, hampered further analysis of the nature of the low Zn phenotypes.

Multimodal synchrotron X-ray fluorescence imaging reveals elemental distribution in seeds and seedlings of the Zn-Cd-Ni hyperaccumulator Noccaea caerulescens.

The molecular biology and genetics of the Ni-Cd-Zn hyperaccumulator Noccaea caerulescens has been extensively studied, but no information is yet available on Ni and Zn redistribution and mobilization during seed germination. Due to the different physiological functions of these elements, and their associated transporter pathways, we expected differential tissue distribution and different modes of translocation of Ni and Zn during germination. This study used synchrotron X-ray fluorescence tomography techniques as well as planar elemental X-ray imaging to elucidate elemental (re)distribution at various stages of the germination process in contrasting accessions of N. caerulescens. The results show that Ni and Zn are both located primarily in the cotyledons of the emerging seedlings and Ni is highest in the ultramafic accessions (up to 0.15 wt%), whereas Zn is highest in the calamine accession (up to 600 µg g-1). The distribution of Ni and Zn in seeds was very similar, and neither element was translocated during germination. The Fe maps were especially useful to obtain spatial reference within the seeds, as it clearly marked the vasculature. This study shows how a multimodal combination of synchrotron techniques can be used to obtain powerful insights about the metal distribution in physically intact seeds and seedlings.

Genetics as a key to improving crop photosynthesis.

Since the basic biochemical mechanisms of photosynthesis are remarkably conserved among plant species, genetic modification approaches have so far been the main route to improve the photosynthetic performance of crops. Yet, phenotypic variation observed in wild species and between varieties of crop species implies there is standing natural genetic variation for photosynthesis, offering a largely unexplored resource to use for breeding crops with improved photosynthesis and higher yields. The reason this has not yet been explored is that the variation probably involves thousands of genes, each contributing only a little to photosynthesis, making them hard to identify without proper phenotyping and genetic tools. This is changing, though, and increasingly studies report on quantitative trait loci for photosynthetic phenotypes. So far, hardly any of these quantitative trait loci have been used in marker assisted breeding or genomic selection approaches to improve crop photosynthesis and yield, and hardly ever have the underlying causal genes been identified. We propose to take the genetics of photosynthesis to a higher level, and identify the genes and alleles nature has used for millions of years to tune photosynthesis to be in line with local environmental conditions. We will need to determine the physiological function of the genes and alleles, and design novel strategies to use this knowledge to improve crop photosynthesis through conventional plant breeding, based on readily available crop plant germplasm. In this work, we present and discuss the genetic methods needed to reveal natural genetic variation, and elaborate on how to apply this to improve crop photosynthesis.

Confined pulsed diffuse layer charging for nanoscale electrodeposition with an STM.

Regulating the state of the solid-liquid interface by means of electric fields is a powerful tool to control electrochemistry. In scanning probe systems, this can be confined closely to a scanning (nano)electrode by means of fast potential pulses, providing a way to probe the interface and control electrochemical reactions locally, as has been demonstrated in nanoscale electrochemical etching. For this purpose, it is important to know the spatial extent of the interaction between pulses applied to the tip, and the substrate. In this paper we use a framework of diffuse layer charging to describe the localization of electrical double layer charging in response to a potential pulse at the probe. Our findings are in good agreement with literature values obtained in electrochemical etching. We show that the pulse can be much more localized by limiting the diffusivity of the ions present in solution, by confined electrodeposition of cobalt in a dimethyl sulfoxide solution, using an electrochemical scanning tunnelling microscope. Finally, we demonstrate the deposition of cobalt nanostructures (<100 nm) using this method. The presented framework therefore provides a general route for predicting and controlling the time-dependent region of interaction between an electrochemical scanning probe and the surface.

Directional quantum dot emission by soft-stamping on silicon Mie resonators.

We present a soft-stamping method to selectively print a homogenous layer of CdSeTe/ZnS core-shell quantum dots (QDs) on top of an array of Si nanocylinders with Mie-type resonant modes. Using this new method, we gain accurate control of the quantum dot's angular emission through engineered coupling of the QDs to these resonant modes. Using numerical simulations we show that the emission into or away from the Si substrate can be precisely controlled by the QD position on the nanocylinder. QDs centered on a 400 nm diameter nanocylinder surface show 98% emission directionality into the Si substrate. Alternatively, for homogenous ensembles placed over the nanocylinder top-surface, the upward emission is enhanced 10-fold for 150 nm diameter cylinders. Experimental PL intensity measurements corroborate the simulated trends with cylinder diameter. PL lifetime measurements reflect well the variations of the local density of states at the QD position due to coupling to the resonant cylinders. These results demonstrate that the soft imprint technique provides a unique manner to directly integrate optical emitters with a wide range of nanophotonic geometries, with potential applications in LEDs, luminescent solar concentrators, and up- and down-conversion schemes for improved photovoltaics.

Isotopic signatures reveal zinc cycling in the natural habitat of hyperaccumulator Dichapetalum gelonioides subspecies from Malaysian Borneo.

BACKGROUND: Some subspecies of Dichapetalum gelonioides are the only tropical woody zinc (Zn)-hyperaccumulator plants described so far and the first Zn hyperaccumulators identified to occur exclusively on non-Zn enriched 'normal' soils. The aim of this study was to investigate Zn cycling in the parent rock-soil-plant interface in the native habitats of hyperaccumulating Dichapetalum gelonioides subspecies (subsp. pilosum and subsp. sumatranum). We measured the Zn isotope ratios (δ66Zn) of Dichapetalum plant material, and associated soil and parent rock materials collected from Sabah (Malaysian Borneo). RESULTS: We found enrichment in heavy Zn isotopes in the topsoil (δ66Zn 0.13 ‰) relative to deep soil (δ66Zn -0.15 ‰) and bedrock (δ66Zn -0.90 ‰). This finding suggests that both weathering and organic matter influenced the Zn isotope pattern in the soil-plant system, with leaf litter cycling contributing significantly to enriched heavier Zn in topsoil. Within the plant, the roots were enriched in heavy Zn isotopes (δ66Zn ~ 0.60 ‰) compared to mature leaves (δ66Zn ~ 0.30 ‰), which suggests highly expressed membrane transporters in these Dichapetalum subspecies preferentially transporting lighter Zn isotopes during root-to-shoot translocation. The shoots, mature leaves and phloem tissues were enriched in heavy Zn isotopes (δ66Zn 0.34-0.70 ‰) relative to young leaves (δ66Zn 0.25 ‰). Thisindicates that phloem sources are enriched in heavy Zn isotopes relative to phloem sinks, likely because of apoplastic retention and compartmentalization in the Dichapetalum subspecies. CONCLUSIONS: The findings of this study reveal Zn cycling in the rock-soil-plant continuum within the natural habitat of Zn hyperaccumulating subspecies of Dichapetalum gelonioides from Malaysian Borneo. This study broadens our understanding of the role of a tropical woody Zn hyperaccumulator plant in local Zn cycling, and highlights the important role of leaf litter recycling in the topsoil Zn budget. Within the plant, phloem plays key role in Zn accumulation and redistribution during growth and development. This study provides an improved understanding of the fate and behaviour of Zn in hyperaccumulator soil-plant systems, and these insights may be applied in the biofortification of crops with Zn.

Quantification of spatial metal accumulation patterns in Noccaea caerulescens by X-ray fluorescence image processing for genetic studies.

BACKGROUND: Hyperaccumulation of trace elements is a rare trait among plants which is being investigated to advance our understanding of the regulation of metal accumulation and applications in phytotechnologies. Noccaea caerulescens (Brassicaceae) is an intensively studied hyperaccumulator model plant capable of attaining extremely high tissue concentrations of zinc and nickel with substantial genetic variation at the population-level. Micro-X-ray Fluorescence spectroscopy (µXRF) mapping is a sensitive high-resolution technique to obtain information of the spatial distribution of the plant metallome in hydrated samples. We used laboratory-based µXRF to characterize a collection of 86 genetically diverse Noccaea caerulescens accessions from across Europe. We developed an image-processing method to segment different plant substructures in the µXRF images. We introduced the concentration quotient (CQ) to quantify spatial patterns of metal accumulation and linked that to genetic variation. RESULTS: Image processing resulted in automated segmentation of µXRF plant images into petiole, leaf margin, leaf interveinal and leaf vasculature substructures. The harmonic means of recall and precision (F1 score) were 0.79, 0.80, 0.67, and 0.68, respectively. Spatial metal accumulation as determined by CQ is highly heritable in Noccaea caerulescens for all substructures, with broad-sense heritability (H2) ranging from 76 to 92%, and correlates only weakly with other heritable traits. Insertion of noise into the image segmentation algorithm barely decreases heritability scores of CQ for the segmented substructures, illustrating the robustness of the trait and the quantification method. Very low heritability was found for CQ if randomly generated substructures were compared, validating the approach. CONCLUSIONS: A strategy for segmenting µXRF images of Noccaea caerulescens is proposed and the concentration quotient is developed to provide a quantitative measure of metal accumulation pattern, which can be used to determine genetic variation for such pattern. The metric is robust to segmentation error and provides reliable H2 estimates. This strategy provides an avenue for quantifying XRF data for analysis of the genetics of metal distribution patterns in plants and the subsequent discovery of new genes that regulate metal homeostasis and sequestration in plants.