Effect of red and blue light supplementation on the efficacy of Noccaea caerulescens in decontaminating metals and alleviating leaching risk.

This study was conducted to investigate the impact of supplementing blue and red light on the biomass yield, metal uptake, contaminant purification, and the alleviation of leaching risks by Noccaea caerulescens, a well-known hyperaccumulator of Cd and Zn. As previously reported for the closely related Thlaspi arvense, N. caerulescens retarded the leaching of Cd and Zn but aggravated the leaching of Pb and Cu, because the species mobilized all metals in soil but only extracted Cd and Zn. Monochromic red light reduced the leaching of Pb and Cu by 13.8% and 1.3%, respectively, but simultaneously weakened Cd phytoremediation by reducing shoot biomass. Our results demonstrated that a small proportion of blue light (10%) could eliminate the negative effect of monochromatic red light on plant shoot growth. However, root biomass decreased by 14.3%, 26.2%, 21.4%, and 61.9% as the percentage of blue light increased from 10 to 100%. Noccaea caerulescens generated the most biomass and accumulated the highest metal concentrations, except for Pb, when the ratio of red to blue light was 1:1. In addition, leachate volume was significantly reduced under the 10% and 50% blue light treatments compared to other light treatments. Therefore, light supplementation with a suitable proportion of blue light can enhance metal purification by N. caerulescens and alleviate potential leaching risk during phytoremediation.

Wide cross-species RNA-Seq comparison reveals convergent molecular mechanisms involved in nickel hyperaccumulation across dicotyledons.

The Anthropocene epoch is associated with the spreading of metals in the environment increasing oxidative and genotoxic stress on organisms. Interestingly, c. 520 plant species growing on metalliferous soils acquired the capacity to accumulate and tolerate a tremendous amount of nickel in their shoots. The wide phylogenetic distribution of these species suggests that nickel hyperaccumulation evolved multiple times independently. However, the exact nature of these mechanisms and whether they have been recruited convergently in distant species is not known. To address these questions, we have developed a cross-species RNA-Seq approach combining differential gene expression analysis and cluster of orthologous group annotation to identify genes linked to nickel hyperaccumulation in distant plant families. Our analysis reveals candidate orthologous genes encoding convergent function involved in nickel hyperaccumulation, including the biosynthesis of specialized metabolites and cell wall organization. Our data also point out that the high expression of IREG/Ferroportin transporters recurrently emerged as a mechanism involved in nickel hyperaccumulation in plants. We further provide genetic evidence in the hyperaccumulator Noccaea caerulescens for the role of the NcIREG2 transporter in nickel sequestration in vacuoles. Our results provide molecular tools to better understand the mechanisms of nickel hyperaccumulation and study their evolution in plants.

Overexpression of ZNT1 and NRAMP4 from the Ni Hyperaccumulator Noccaea caerulescens Population Monte Prinzera in Arabidopsis thaliana Perturbs Fe, Mn, and Ni Accumulation.

Metalliferous soils are characterized by a high content of metal compounds that can hamper plant growth. The pseudometallophyte Noccaea caerulescens is able to grow on metalliferous substrates by implementing both tolerance and accumulation of usually toxic metal ions. Expression of particular transmembrane transporter proteins (e.g., members of the ZIP and NRAMP families) leads to metal tolerance and accumulation, and its comparison between hyperaccumulator N. caerulescens with non-accumulator relatives Arabidopsis thaliana and Thlaspi arvense has deepened our knowledge on mechanisms adopted by plants to survive in metalliferous soils. In this work, two transporters, ZNT1 and NRAMP4, expressed in a serpentinic population of N. caerulescens identified on the Monte Prinzera (Italy) are considered, and their expression has been induced in yeast and in A. thaliana. In the latter, single transgenic lines were crossed to test the effect of the combined over-expression of the two transporters. An enhanced iron and manganese translocation towards the shoot was induced by overexpression of NcZNT1. The combined overexpression of NcZNT1 and NcNRAMP4 did perturb the metal accumulation in plants.

Transcriptional effects of cadmium on iron homeostasis differ in calamine accessions of Noccaea caerulescens.

Calamine accessions of the zinc/cadmium/nickel hyperaccumulator, Noccaea caerulescens, exhibit striking variation in foliar cadmium accumulation in nature. The Ganges accession (GA) from Southern France displays foliar cadmium hyperaccumulation (>1000 µg g-1 DW), whereas the accession La Calamine (LC) from Belgium, with similar local soil metal composition, does not (<100 µg g-1 DW). All calamine accessions are cadmium hypertolerant. To find out the differences between LC and GA in their basic adaptation mechanisms, we bypassed the cadmium excluding phenotype of LC by exposing the plants to 50 µm cadmium in hydroponics, achieving equal cadmium accumulation in the shoots. The iron content increased in the roots of both accessions. GA exhibited significant decreases in manganese and zinc contents in the roots and shoots, approaching those in LC. Altogether 702 genes responded differently to cadmium exposure between the accessions, 157 and 545 in the roots and shoots, respectively. Cadmium-exposed LC showed a stress response and had decreased levels of a wide range of photosynthesis-related transcripts. GA showed less changes, mainly exhibiting an iron deficiency-like response. This included increased expression of genes encoding five iron deficiency-regulated bHLH transcription factors, ferric reduction oxidase FRO2, iron transporters IRT1 and OPT3, and nicotianamine synthase NAS1, and decreased expression of genes encoding ferritins and NEET (a NEET family iron-sulfur protein), which is possibly involved in iron transfer, distribution and/or management. The function of the IRT1 gene in the accessions was compared. We conclude that the major difference between the two accessions is in the way they cope with iron under cadmium exposure.

A new insight on structural and some functional aspects of peri-endodermal thickenings, a specific layer in Noccaea caerulescens roots.

BACKGROUND AND AIMS: Cell walls of the peri-endodermis, a layer adjacent to the endodermis in alpine pennycress (Noccaea caerulescens) roots, form C-shaped peri-endodermal thickenings (PETs). Despite its specific position close to the endodermis, the assumed similarity of PETs to phi thickenings in many other species, and the fact that N. caerulescens is a well-studied heavy-metal-hyperaccumulating plant, the PET as a root trait is still not understood. METHODS: Here, we characterized PET cell walls by histochemical techniques, Raman spectroscopy, immunolabelling and electron microscopy. Moreover, a role of PETs in solute transport was tested and compared with Arabidopsis thaliana plants, which do not form PETs in roots. KEY RESULTS: Cell walls with PETs have a structured relief mainly composed of cellulose and lignin. Suberin, typical of endodermal cells, is missing but pectins are present on the inner surface of the PET. Penetrating dyes are not able to cross PETs either by the apoplasmic or the symplasmic pathway, and a significantly higher content of metals is found in root tissues outside of PETs than in innermost tissues. CONCLUSIONS: Based on their development and chemical composition, PETs are different from the endodermis and closely resemble phi thickenings. Contrarily, the different structure and dye impermeability of PETs, not known in the case of phi thickenings, point to an additional barrier function which makes the peri-endodermis with PETs a unique and rare layer.

Identification of Glucosinolates in Seeds of Three Brassicaceae Species Known to Hyperaccumulate Heavy Metals.

Plants from the Brassicaceae family are known to contain secondary metabolites called glucosinolates. Our goal was to establish by LC/MS the glucosinolate profile of seeds of three Brassicaceae species known to hyperaccumulate heavy metals. We investigated Alyssum fallacinum auct. non Hausskn., Iberis intermedia Guers., and Noccaea caerulescens (J. Presl & C. Presl) F. K. Mey. Our results indicate that A. fallacinum seeds contain glucoiberin and glucoibervirin, which had not been previously identified in this plant. Furthermore, we report for the first time the presence of glucoiberin, glucoibervirin, glucotropaeolin, and sinigrin in I. intermedia. We have detected for the first time glucoconringiin in N. caerulescens. In addition, glucosinalbin, 4-hydroxyglucobrassicin, and glucomoringin were also detected.

Lower selfing rates in metallicolous populations than in non-metallicolous populations of the pseudometallophyte Noccaea caerulescens (Brassicaceae) in Southern France.

BACKGROUND AND AIMS: The pseudometallophyte Noccaea caerulescens is an excellent model to study evolutionary processes, as it grows both on normal and on heavy-metal-rich, toxic soils. The evolution and demography of populations are critically impacted by mating system and, yet, information about the N. caerulescens mating system is limited. METHODS: Mean selfing rates were assessed using microsatellite loci and a robust estimation method (RMES) in five metallicolous and five non-metallicolous populations of N. caerulescens in Southern France, and this measure was replicated for two successive reproductive seasons. As a part of the study, the patterns of gene flow among populations were analysed. The mating system was then characterized at a fine spatial scale in three populations using the MLTR method on progeny arrays. KEY RESULTS: The results confirm that N. caerulescens has a mixed mating system, with selfing rates ranging from 0·2 to 0·5. Selfing rates did not vary much among populations within ecotypes, but were lower in the metallicolous than in the non-metallicolous ecotype, in both seasons. Effective population size was also lower in non-metallicolous populations. Biparental inbreeding was null to moderate. Differentiation among populations was generally high, but neither ecotype nor isolation by distance explained it. CONCLUSIONS: The consequences of higher selfing rates on adaptation are expected to be weak to moderate in non-metallicolous populations and they are expected to suffer less from inbreeding depression, compared to metallicolous populations.

Root and shoot transcriptome analysis of two ecotypes of Noccaea caerulescens uncovers the role of NcNramp1 in Cd hyperaccumulation.

The Zn/Cd hyperaccumulator, Noccaea caerulescens, has been studied extensively for its ability to accumulate high levels of Zn and Cd in its leaves. Previous studies have indicated that the Zn and Cd hyperaccumulation trait exhibited by this species involves different transport and tolerance mechanisms. It has also been well documented that certain ecotypes of N. caerulescens are much better Cd hyperaccumulators than others. However, there does not seem to be much ecotypic variation for Zn hyperaccumulation in N. caerulescens. In this study we employed a comparative transcriptomics approach to look at root and shoot gene expression in Ganges and Prayon plants in response to Cd stress to identify transporter genes that were more highly expressed in either the roots or shoots of the superior Cd accumulator, Ganges. Comparison of the transcriptomes from the two ecotypes of Noccaea caerulescens identified a number of genes that encoded metal transporters that were more highly expressed in the Ganges ecotype in response to Cd stress. Characterization of one of these transporters, NcNramp1, showed that it is involved in the influx of Cd across the endodermal plasma membrane and thus may play a key role in Cd flux into the stele and root-to-shoot Cd transport. NcNramp1 may be one of the main transporters involved in Cd hyperaccumulation in N. caerulescens and copy number variation appears to be the main reason for high NcNramp1 gene expression underlying the increased Cd accumulation in the Ganges ecotype.

A proteomics approach to investigate the process of Zn hyperaccumulation in Noccaea caerulescens (J & C. Presl) F.K. Meyer.

Zinc (Zn) is an essential trace element in all living organisms, but is toxic in excess. Several plant species are able to accumulate Zn at extraordinarily high concentrations in the leaf epidermis without showing any toxicity symptoms. However, the molecular mechanisms of this phenomenon are still poorly understood. A state-of-the-art quantitative 2D liquid chromatography/tandem mass spectrometry (2D-LC-MS/MS) proteomics approach was used to investigate the abundance of proteins involved in Zn hyperaccumulation in leaf epidermal and mesophyll tissues of Noccaea caerulescens. Furthermore, the Zn speciation in planta was analyzed by a size-exclusion chromatography/inductively coupled plasma mass spectrometer (SEC-ICP-MS) method, in order to identify the Zn-binding ligands and mechanisms responsible for Zn hyperaccumulation. Epidermal cells have an increased capability to cope with the oxidative stress that results from excess Zn, as indicated by a higher abundance of glutathione S-transferase proteins. A Zn importer of the ZIP family was more abundant in the epidermal tissue than in the mesophyll tissue, but the vacuolar Zn transporter MTP1 was equally distributed. Almost all of the Zn located in the mesophyll was stored as Zn-nicotianamine complexes. In contrast, a much lower proportion of the Zn was found as Zn-nicotianamine complexes in the epidermis. However, these cells have higher concentrations of malate and citrate, and these organic acids are probably responsible for complexation of most epidermal Zn. Here we provide evidence for a cell type-specific adaptation to excess Zn conditions and an increased ability to transport Zn into the epidermal vacuoles.

Histidine-mediated xylem loading of zinc is a species-wide character in Noccaea caerulescens.

Histidine plays a crucial role in nickel (Ni) translocation in Ni-hyperaccumulating plants. Here, we investigated its role in zinc (Zn) translocation in four accessions of the Zn hyperaccumulator, Noccaea caerulescens, using the related non-hyperaccumulator, Thlaspi arvense, as a reference. We compared the effects of exogenous histidine supply on Zn xylem loading, and of Zn-histidine complex formation on Zn uptake in energized tonoplast vesicles. The Zn distribution patterns over root tissues were also compared. Exogenous histidine supply enhanced Zn xylem loading in all the N. caerulescens accessions, but decreased it in T. arvense. Zn distribution patterns over root tissues were similar, apart from the accumulation in cortical and endodermal cells, which was much lower in N. caerulescens than in T. arvense. Zn uptake in energized tonoplast vesicles was inhibited significantly in N. caerulescens, but not affected significantly in T. arvense, when Zn was supplied in combination with histidine in a 1:2 molar ratio. Histidine-mediated Zn xylem loading seems to be a species-wide character in N. caerulescens. It may well have evolved as a component trait of the hyperaccumulation machinery for Zn, rather than for Ni.

Uncoupling of reactive oxygen species accumulation and defence signalling in the metal hyperaccumulator plant Noccaea caerulescens.

The metal hyperaccumulator plant Noccaea caerulescens is protected from disease by the accumulation of high concentrations of metals in its aerial tissues, which are toxic to many pathogens. As these metals can lead to the production of damaging reactive oxygen species (ROS), metal hyperaccumulator plants have developed highly effective ROS tolerance mechanisms, which might quench ROS-based signals. We therefore investigated whether metal accumulation alters defence signalling via ROS in this plant. We studied the effect of zinc (Zn) accumulation by N. caerulescens on pathogen-induced ROS production, salicylic acid accumulation and downstream defence responses, such as callose deposition and pathogenesis-related (PR) gene expression, to the bacterial pathogen Pseudomonas syringae pv. maculicola. The accumulation of Zn caused increased superoxide production in N. caerulescens, but inoculation with P. syringae did not elicit the defensive oxidative burst typical of most plants. Defences dependent on signalling through ROS (callose and PR gene expression) were also modified or absent in N. caerulescens, whereas salicylic acid production in response to infection was retained. These observations suggest that metal hyperaccumulation is incompatible with defence signalling through ROS and that, as metal hyperaccumulation became effective as a form of elemental defence, normal defence responses became progressively uncoupled from ROS signalling in N. caerulescens.

Maximizing trace metal phytoextraction through planting methods: Role of rhizosphere fertility and microbial activities.

Brownfields are a widespread problem in the world. The poor quality of these soils and the potential presence of contaminants can pose a significant threat to plant establishment and growth. However, it may be possible to improve their establishment with an appropriate agricultural practice. In this paper, the effects of two common planting strategies, seeding and transplanting, on the establishment and growth of the hyperaccumulator species Noccaea caerulescens and on its phytoextraction capacity were investigated. A field experiment was conducted by direct sowing of N. caerulescens seeds on a plot of contaminated Technosols in Jeandelaincourt, France. At the same time, seeds were sown on potting soil under controlled conditions. One month later, the seedlings were transplanted to the field. One year later, the results showed that transplanting improved the establishment and growth of N. caerulescens. This was due to a decrease in soil pH in the rhizosphere, which subsequently increased nutrient availability. This change in rhizosphere properties also appeared to be the key that improved microbial activities in the rhizosphere soil of transplanted plants. The observed improvement in both rhizosphere nutrient availability and microbial activities, in turn, increased auxin concentrations in the rhizosphere and consequently a more developed root system was observed in the transplanted plants. Furthermore, the Cd and Zn phytoextraction yield of transplanted plants is 2.5 and 5 times higher, respectively, than that of sown plants. In conclusion, N. caerulescens transplantation on contaminated sites seems to be an adequate strategy to improve plant growth and enhance trace metal phytoextraction.

Metal Interactions in the Ni Hyperaccumulating Population of Noccaea caerulescens Monte Prinzera.

Hyperaccumulation is a fascinating trait displayed by a few plant species able to accumulate large amounts of metal ions in above-ground tissues without symptoms of toxicity. Noccaea caerulescens is a recognized model system to study metal hyperaccumulation and hypertolerance. A N. caerulescens population naturally growing on a serpentine soil in the Italian Apennine Mountains, Monte Prinzera, was chosen for the study here reported. Plants were grown hydroponically and treated with different metals, in excess or limiting concentrations. Accumulated metals were quantified in shoots and roots by means of ICP-MS. By real-time PCR analysis, the expression of metal transporters and Fe deficiency-regulated genes was compared in the shoots and roots of treated plants. N. caerulescens Monte Prinzera confirmed its ability to hypertolerate and hyperaccumulate Ni but not Zn. Moreover, excess Ni does not induce Fe deficiency as in Ni-sensitive species and instead competes with Fe translocation rather than its uptake.

Endophyte Community Changes in the Seeds of Eight Plant Species following Inoculation with a Multi-Endophytic Bacterial Consortium and an Individual Sphingomonas wittichii Strain Obtained from Noccaea caerulescens.

Noccaea caerulescens, a hyperaccumulator plant species known for its metal tolerance and accumulation abilities, harbours a microbiome of interest within its seed. These seed-associated bacteria, often referred to as seed endophytes, play a unique role in seed germination and plant growth and health. This work aimed to address how inoculating seeds of eight different plant species-Medicago sativa (alfalfa), Zea mays (corn), Raphanus sativus (radish), Helianthus annus (sunflower), Cucurbita pepo subsp. pepo (squash), Beta vulgaris subsp. cicla (rainbow chard), Arabidopsis thaliana (thale cress), and Noccaea caerulescens (penny cress)-with a bacterial consortium made from the seed endophytes of N. caerulescens would affect the seed microbiome of each test plant species, as well as inoculation with a strain of the bacterium Sphingomonas wittichii, which was previously isolated from seeds of N. caerulescens. Additionally, we aimed to offer preliminary plant tests in order to determine the best seed treatment plan for future research. The results showed that inoculation with the bacterial consortium held the most potential for increasing plant size (p < 0.001) and increasing germination rate (p < 0.05). The plant that responded best to inoculation was N. caerulescens (penny cress), likely because the microbes being introduced into the seed were not foreign. This paper also offers the first insight into the seed endophytes of Beta vulgaris subsp. cicla, highlighting an abundance of Proteobacteria, Firmicutes, and Actinobacteriota.

Community Profiling of Seed Endophytes from the Pb-Zn Hyperaccumulator Noccaea caerulescens and Their Plant Growth Promotion Potential.

Endophytes within plants are known to be crucial for plant fitness, and while their presence and functions in many compartments have been studied in depth, the research on seed endophytes is still limited. This work aimed to characterize the seed endophytic and rhizospheric bacterial community of two Noccaea caerulescens Pb-Zn hyperaccumulator populations, growing on two heavy-metal-polluted sites in Belgium. Cultured representatives were evaluated for their potential to enhance seed germination and root length of the model species Arabidopsis thaliana. The results indicated that the community structure within the seed is conserved between the two locations, comprising mainly of Proteobacteria (seeds), and Actinobacteria in the bulk soil. Root length of A. thaliana was significantly increased when inoculated with Sphingomonas vulcanisoli. The results of this paper offer insights into the importance of the selection of the core seed endophytic microbiome and highlight the precarious symbiotic relationship they have with the plant and seed.

Cadmium and Zn hyperaccumulation provide efficient constitutive defense against Turnip yellow mosaic virus infection in Noccaea caerulescens.

To understand the role of Zn and Cd in anti-viral defence, Zn/Cd hyperaccumulator Noccaea caerulescens plants grown with deficient (0.3 µM), replete (10 µM) and excess (100 µM) Zn2+ and Cd (10 µM Zn2+ + 1 µM Cd2+) were infected with Turnip yellow mosaic virus (TYMV). Gas exchange and chlorophyll fluorescence kinetics analyses demonstrated direct TYMV effects on photosynthetic light reactions but N. caerulescens was more resistant against TYMV than the previously studied non-hyperaccumulator N. ochroleucum. Virus abundance and photosynthesis inhibition were the lowest in the high Zn and Cd treatments. RNAseq analysis of 10 µM Zn2+ plants revealed TYMV-induced upregulation of Ca transporters, chloroplastic ZTP29 and defence genes, but none of those that are known to be strongly involved in hyperaccumulation. Synchrotron µ-XRF tomography, however, showed that Zn hyperaccumulation remained strongest in vacuoles of epidermal storage cells regardless of infection. This was in contrast to N. ochroleucum, where apoplastic Zn drastically increased in response to TYMV. These results suggest that the antiviral response of N. caerulescens is less induced by the onset of this biotic stress, but it is rather a permanent resistant state of the plant. Real-time qPCR revealed upregulation of ferritin in Zn10 infected plants, suggesting Fe deprivation as a virus defence strategy under suboptimal Zn supply.

DNA methylation is enhanced during Cd hyperaccumulation in Noccaea caerulescens ecotype Ganges.

In this study, we assess the DNA damage occurring in response to cadmium (Cd) in the Cd hyperaccumulator Noccaea caerulescens Ganges (GA) vs the non-accumulator and close-relative species Arabidopsis thaliana. At this purpose, the alkaline comet assay was utilized to evaluate the Cd-induced variations in nucleoids and the methy-sens comet assay, and semiquantitative real-time (qRT)-PCR were also performed to associate nucleus variations to possible DNA modifications. Cadmium induced high DNA damages in nuclei of A. thaliana while only a small increase in DNA migration was observed in N. caerulescens GA. In addition, in N. caerulescens GA, CpG DNA methylation increase upon Cd when compared to control condition, along with an increase in the expression of MET1 gene, coding for the DNA-methyltransferase. N. caerulescens GA does not show any oxidative stress under Cd treatment, while A. thaliana Cd-treated plants showed an upregulation of transcripts of the respiratory burst oxidase, accumulation of reactive oxygen species, and enhanced superoxide dismutase activity. These data suggest that epigenetic modifications occur in the N. caerulescens GA exposed to Cd to preserve genome integrity, contributing to Cd tolerance.

[Uptake and Accumulation of Cadmium and Zinc by Two Energy Grasses: A Field Experiment].

The remediation potential of large biomass energy grasses in cadmium-contaminated soil remains ambiguous. A field experiment was carried out in a cadmium-contaminated farmland using two energy grasses and two control plants. The two energy grasses were hybrid pennisetum (Pennisetum americanum×P. purpureum, PAP) and purple elephant grass (P. purpureum 'Purple', PPP), and the two control plants were Iris lactea var. chinensis (ILC) and a cadmium hyperaccumulator, Noccaea caerulescens (NC). The results showed that the aboveground biomass of PAP was the largest among the four plants, and 126 and 36 times that of NC and ILC, respectively, but no significant difference with that of PPP. The concentrations of cadmium and zinc in the shoots and roots of NC were significantly higher than in the other plants. Zinc concentrations in the shoots and roots of ILC were lower than in the other plants, while cadmium concentrations were significantly higher than in PAP and PPP (P<0.05). The amounts of cadmium and zinc accumulated in the shoots of PPP were the highest among the four plants, while cadmium concentrations in the shoots and roots of PPP were significantly lower than in ILC and NC (P<0.05). Cadmium amounts accumulated in PPP shoots were 7.0 and 4.1 times that of ILC and NC, respectively. Zinc amounts accumulated in PPP shoots were 41 and 11 times that of ILC and NC, respectively (P<0.05). Cadmium accumulation in the shoots of PAP was 19.4% lower than in PPP, and zinc accumulation had no significant difference with that of PPP. NC, having a bioconcentration factor of shoot (BCFS) and a translocation factor (TF) for cadmium and zinc both larger than 1, is usable for phytoextraction of soils contaminated by cadmium and zinc. ILC, having a bioconcentration factor of root (BCFR) larger than 1 and a TF lower than 1 for cadmium, is usable for the phytostabilization of soils contaminated by cadmium. PPP, having a BCFR larger than 1 and a TF lower than 1 for zinc, can be used in the phytostabilization of soils contaminated by zinc. Under field conditions, PPP and PAP showed great potential for the extraction and removal of cadmium and zinc from soil due to their large biomass and ability to produce economic benefits, have good application prospects.

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.

The influence of light combination on the physicochemical characteristics and enzymatic activity of soil with multi-metal pollution in phytoremediation.

Light irradiation with suitable quality and intensity could influence the success of phytoremediation by improving the biomass yield of plants. However, mechanisms involved in this influence on the contaminant accumulation and translocation ability of plants have rarely been studied. Five light combinations with different red (R) and blue (B) ratios (0, 10, 50, 75 and 100 % blue) at the same intensity (220 µmol m-2 s-1) were used to assist phytoremediation using Noccaea caerulescens, and the change in physicochemical characteristics and enzymatic activities of soils after phytoremediation were evaluated. Compared with the control, the light combinations and monochromic blue light significantly increased the activities of soil ureases, invertases, and phosphatases, whereas monochromic red light strongly inhibited the activities of these enzymes, because different light irradiations altered the formation and excretion of carbohydrates from plants for soil microorganism consumption. Plants under B50R50 treatment accumulated the highest concentrations of metals, but their chlorophyll concentrations and lipid peroxidation were similar to those other species with lower metal concentrations. Hence, light with a proper blue/red ratio can simultaneously improve the physicochemical characteristics and enzymatic activities of soils, increase the metal uptake capacity and oxidation resistance of plants, and reduce the leaching risk during phytoremediation processes.