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.

The rhizosphere microbiota of the zinc and cadmium hyperaccumulators Arabidopsis halleri and Noccaea caerulescens is highly convergent in Prayon (Belgium)

The Prayon site is known as a zinc-polluted area where two zinc and cadmium hyperaccumulator plant species currently coexist, although Arabidopsis halleri was introduced more recently than Noccaea caerulescens . While soil microorganisms may influence metal uptake, the microbial community present in the rhizosphere of hyperaccumulators remains poorly known. Plants of both species were sampled with their bulk and rhizosphere soil from different plots of the Prayon site. Soil components (ionome, pH, water composition, temperature) were analyzed, as well as shoot ionome and expression levels of metal transporter genes ( HMA3 , HMA4 , ZIP4 / ZNT1 , ZIP6 , MTP1 ). The taxonomic diversity of the microorganisms in soil samples was then determined by 16S rRNA metabarcoding and compared at the Operational Taxonomy Unit (OTU) level and across different taxonomic levels. Our elemental analyses confirmed that the site is still highly contaminated with zinc and cadmium and that both plant species indeed hyperaccumulate these elements in situ . Although the pollution is overall high, it is heterogenous at the site scale and correlates with the expression of some metal transporter genes. Metabarcoding analyses revealed a decreasing gradient of microbial diversity, with more OTUs discovered in the rhizosphere than in the soil bulk, especially at the bottom of the cores. However, the variability gradient increases with the distance from roots. Using an ad hoc pseudo-taxonomy to bypass the biases caused by a high proportion of unclassified and unknown OTUs, we identified Chloroflexi, Armatimonadetes, Pirellulaceae, Gemmatimonadetes and Chitinophagaceae as the drivers of the differences in the gradient along the cores. In contrast, no significant difference was identified between the rhizosphere composition of A. halleri and N. caerulescens . This suggests that, despite their distinct colonization history in Prayon, the two plant species have now recruited highly convergent microbial communities in the rhizosphere.

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.

Natural Fungal Endophytes From Noccaea caerulescens Mediate Neutral to Positive Effects on Plant Biomass, Mineral Nutrition and Zn Phytoextraction

Phytoextraction using hyperaccumulating plants is a method for the remediation of soils contaminated with trace elements (TEs). As a strategy for improvement, the concept of fungal-assisted phytoextraction has emerged in the last decade. However, the role played by fungal endophytes of hyperaccumulating plants in phytoextraction is poorly studied. Here, fungal endophytes isolated from calamine or non-metalliferous populations of the Cd/Zn hyperaccumulator Noccaea caerulescens were tested for their growth promotion abilities affecting the host plant. Plants were inoculated with seven different isolates and grown for 2 months in trace element (TE)-contaminated soil. The outcomes of the interactions between N. caerulescens and its native strains ranged from neutral to beneficial. Among the strains, Alternaria thlaspis and Metapochonia rubescens, respectively, isolated from the roots of a non-metallicolous and a calamine population of N. caerulescens, respectively, exhibited the most promising abilities to enhance the Zn phytoextraction potential of N. caerulescens related to a significant increase of the plant biomass. These strains significantly increased the root elemental composition, particularly in the case of K, P, and S, suggesting an improvement of the plant nutrition. Results obtained in this study provide new insights into the relevance of microbial-assisted phytoextraction approaches in the case of hyperaccumulating plants.