Image_1_Cellular Fractionation and Nanoscopic X-Ray Fluorescence Imaging Analyses Reveal Changes of Zinc Distribution in Leaf Cells of Iron-Deficient Plants.TIF

<p>Multilevel interactions among nutrients occur in the soil-plant system. Among them, Fe and Zn homeostasis in plants are of great relevance because of their importance for plant and human nutrition. However, the mechanisms underlying the interplay between Fe and Zn in plants are still poorly understood. In order to elucidate how Zn interacts with Fe homeostasis, it is crucial to assess Zn distribution either in the plant tissues or within the cells. In this study, we investigated the subcellular Zn distribution in Fe-deficient leaf cells of cucumber plants by using two different approaches: cellular fractionation coupled with inductively coupled plasma mass spectrometry (ICP/MS) and nanoscopic synchrotron X-ray fluorescence imaging. Fe-deficient leaves showed a strong accumulation of Zn as well as a strong alteration of the organelles’ ultrastructure at the cellular level. The cellular fractionation-ICP/MS approach revealed that Zn accumulates in both chloroplasts and mitochondria of Fe deficient leaves. Nano-XRF imaging revealed Zn accumulation in chloroplast and mitochondrial compartments, with a higher concentration in chloroplasts. Such results show that (i) both approaches are suitable to investigate Zn distribution at the subcellular level and (ii) cellular Fe and Zn interactions take place mainly in the organelles, especially in the chloroplasts.</p

Image_3_Cellular Fractionation and Nanoscopic X-Ray Fluorescence Imaging Analyses Reveal Changes of Zinc Distribution in Leaf Cells of Iron-Deficient Plants.pdf

<p>Multilevel interactions among nutrients occur in the soil-plant system. Among them, Fe and Zn homeostasis in plants are of great relevance because of their importance for plant and human nutrition. However, the mechanisms underlying the interplay between Fe and Zn in plants are still poorly understood. In order to elucidate how Zn interacts with Fe homeostasis, it is crucial to assess Zn distribution either in the plant tissues or within the cells. In this study, we investigated the subcellular Zn distribution in Fe-deficient leaf cells of cucumber plants by using two different approaches: cellular fractionation coupled with inductively coupled plasma mass spectrometry (ICP/MS) and nanoscopic synchrotron X-ray fluorescence imaging. Fe-deficient leaves showed a strong accumulation of Zn as well as a strong alteration of the organelles’ ultrastructure at the cellular level. The cellular fractionation-ICP/MS approach revealed that Zn accumulates in both chloroplasts and mitochondria of Fe deficient leaves. Nano-XRF imaging revealed Zn accumulation in chloroplast and mitochondrial compartments, with a higher concentration in chloroplasts. Such results show that (i) both approaches are suitable to investigate Zn distribution at the subcellular level and (ii) cellular Fe and Zn interactions take place mainly in the organelles, especially in the chloroplasts.</p

Three-Dimensional Reconstruction, by TEM Tomography, of the Ultrastructural Modifications Occurring in <i>Cucumis sativus</i> L. Mitochondria under Fe Deficiency

<div><p>Background</p><p>Mitochondria, as recently suggested, might be involved in iron sensing and signalling pathways in plant cells. For a better understanding of the role of these organelles in mediating the Fe deficiency responses in plant cells, it is crucial to provide a full overview of their modifications occurring under Fe-limited conditions. The aim of this work is to characterize the ultrastructural as well as the biochemical changes occurring in leaf mitochondria of cucumber (<i>Cucumis sativus</i> L.) plants grown under Fe deficiency.</p><p>Methodology/Results</p><p>Mitochondrial ultrastructure was investigated by transmission electron microscopy (TEM) and electron tomography techniques, which allowed a three-dimensional (3D) reconstruction of cellular structures. These analyses reveal that mitochondria isolated from cucumber leaves appear in the <i>cristae junction model</i> conformation and that Fe deficiency strongly alters both the number and the volume of cristae. The ultrastructural changes observed in mitochondria isolated from Fe-deficient leaves reflect a metabolic status characterized by a respiratory chain operating at a lower rate (orthodox-like conformation) with respect to mitochondria from control leaves.</p><p>Conclusions</p><p>To our knowledge, this is the first report showing a 3D reconstruction of plant mitochondria. Furthermore, these results suggest that a detailed characterization of the link between changes in the ultrastructure and functionality of mitochondria during different nutritional conditions, can provide a successful approach to understand the role of these organelles in the plant response to Fe deficiency.</p></div

Mitochondrial functionality of leaves from cucumber plants grown in control conditions (C) or under Fe deficiency (-Fe).

<p><b>(A)</b><i>In vivo</i> O₂ consumption rates expressed as μmol O₂ consumed min^-1 g FW^-1. The total O₂ consumption rate (IR), as well as the residual O₂ consumption, measured by using KCN (inhibiting cytochrome c oxidase) (IR + KCN) or KCN in combination with SHAM (inhibiting alternative oxidase) (IR + KCN +SHAM) were measured. Values represent mean ±SE of three independent measurements. <b>(B)</b> For each growth condition, the residual O₂ consumption (IR +KCN+SHAM), reported in (A), is shown as % value, with respect to correspondent IR. Mitochondrial respiration is the difference between IR and residual O₂ consumption. Student t-test was used to analyse statistical significance with respect to controls. *:p<0,05; **:p<0,01; ***:p<0,001. <b>(C)</b> Mitochondria (M) and chloroplasts (Cp) were purified from control plants by percoll gradient; the Cp and M fractions at 35/80% percoll and 23/40% percoll layers interfaces, respectively, are indicated with arrows in the gradient tubes. The purified Cp and M fractions were tested by western blot, by using antibodies against two protein markers: Toc33 and porin, specific for Cp, and M, respectively. 10 μg total proteins were loaded in each lane. <b>(D)</b> Western blot analysis of three different mitochondrial Fe-containing proteins (AOX, Rieske, cyt c) on mitochondria purified from leaves of control (C) or Fe-deficient (-Fe) plants. Porin was used as loading control; 10 μg total proteins were loaded in each lane. The results are representative of tree independent experiments.</p

Three-dimensional models of leaf mitochondria from cucumber plants grown in control condition (C) or under Fe deficiency (-Fe).

<p>Different colours were used for the rendering of the different suborganellar structures: magenta for inner membranes (IM), blue for outer membranes (OM), green for cristae and red for cristae junctions. <b>(A-D)</b> Tomographic slices of mitochondria from control (A,B) and Fe-deficient (C,D) plants; the inner membrane (IM), the outer membrane (OM), and the matrix enclosed by the IM are indicated; in particular, the rendering of the mitochondria structures from control (B) and Fe-deficient (D) plants, superimposed on the tomographic slices, is reported. <b>(E,F)</b> Three-dimensional models of the mitochondria from control (E) or Fe-deficient (F) plants. <b>(G,H)</b> Details of mitochondrial cristae junctions, in red, identified in mitochondrial from control (G) or Fe-deficient (H) plants. Scale bars 100 nm.</p

Physiological characterization of leaves from cucumber plants grown in control condition (C) or under Fe deficiency (–Fe).

<p><b>(A)</b> Phenotype of 10 days old cucumber plants grown under control condition (C) or under Fe deficiency (-Fe). The following parameters have been evaluated, on expanded leaves of plants grown as in (A): <b>(B)</b> total chlorophyll concentration, expressed as mg chlorophyll g FW^-1; <b>(C)</b> O₂ evolution, expressed as μmol O₂ min^-1 g FW^-1; <b>(D)</b> net photosynthesis expressed as μmol CO₂ consumed s^-1 m^-2; <b>(E)</b> evapotranspiration, expressed as mmol H₂O s^-1m^-2; <b>(F)</b> stomatal conductance, expressed as mmol CO₂ s^-1m^-2; <b>(G)</b> manganese (Mn), iron (Fe), copper (Cu), zinc (Zn), molybdenum (Mo) concentrations, expressed as μg g DW^-1. Data are means ± SE of at least three independent experiments. Student t-test was used to analyse statistical significance with respect to controls. *:p<0,05; **:p<0,01; ***:p<0,001.</p

Determination of the cristae number <i>per</i> mitochondrion and the relative intracristae surface area (intracristae surface area <i>per</i> mitochondrion surface area, expressed as % ratio).

<p>Mean values ± SD are from analysis of one hundred mitochondria randomly selected from inclusions obtained from three independent biological samples. Student t-test was used to analyse statistical significance with respect to controls.</p><p>***:p<0,001</p><p>Determination of the cristae number <i>per</i> mitochondrion and the relative intracristae surface area (intracristae surface area <i>per</i> mitochondrion surface area, expressed as % ratio).</p

Transmission electron microscopy (TEM) analysis of leaf tissues from cucumber plants grown in control condition (C) or under Fe deficiency (-Fe).

<p>Control leaf tissues (<b>A-C</b>) and -Fe leaf tissues (<b>D-F</b>). All scale bars = 500 nm. M: mitochondria; C: chloroplast; V: vacuole.</p

Data for "The mycorrhizal root-shoot axis elicits Coffea arabica growth under low phosphate conditions"

This folder contains relevant datasets generated in the study: Chialva M., Patono D.L, de Souza L.P., Novero M., Vercellino S., Maghrebi M., Morgante M., Lovisolo C., Vigani G., Fernie A., Fiorilli V., Lanfranco L., Bonfante P. (2023) The mycorrhizal root-shoot axis elicits Coffea arabica growth under low phosphate conditions. The New Phytol, in press. RNA-seq dataset and annotations RNAseq_raw_counts.tsv > Raw mRNA-seq reads counts obtained by pseudo-aligning reads on the C. arabica reference transcriptome (NCBI reference genome accession GCF_003713225.1) using Salmon software in selective alignment mode and summarized using 'tximport' library in R. interproscan_proteins.local.tsv > the parsed InterProScan analysis output run on known Coffea arabica proteins (NCBI reference genome accession GCF_003713225.1). gene2ko.tsv > Protein IDs to Kegg Orthologs (KO) IDs obtained using KAAS server. Orthogroups inference Orthogroups predicted with Orthofinder (v.2.5.4) on Coffea arabica, C. canephora, C. eugenioides, S. lycopersicum, P. trichocarpa, M. truncatula, and Vitis vinifera proteomes HOGs_N0.tsv > Phylogenetic Hierachical Orthogroups (HOGs) predicted by Orthofinder on proteomes at N0 hierachical level Orthogroups.tsv > Orthogroups predicted by Orthofinder (v.2.5.4) on proteomes (old-way) OGs.tsv > refined OGs used in this study (manually integrating some missing genes) Metabolomics Metabolites_DB.xlsx > An exhaustive list of detected coffee metabolites in previous studies used to enhance annotation of LC-MS spectra generated in this study. Note: Sample names are coded by condition (M or MYC=mycorrhizal plants, C or NM=non-mycorrhized controls) and by organ type (s=leaves, r=roots)</p

A Drought Resistance-Promoting Microbiome Is Selected by Root System under Desert Farming

<div><h3>Background</h3><p>Traditional agro-systems in arid areas are a bulwark for preserving soil stability and fertility, in the sight of “reverse desertification”. Nevertheless, the impact of desert farming practices on the diversity and abundance of the plant associated microbiome is poorly characterized, including its functional role in supporting plant development under drought stress.</p> <h3>Methodology/Principal Findings</h3><p>We assessed the structure of the microbiome associated to the drought-sensitive pepper plant (<em>Capsicum annuum</em> L.) cultivated in a traditional Egyptian farm, focusing on microbe contribution to a crucial ecosystem service, i.e. plant growth under water deficit. The root system was dissected by sampling root/soil with a different degree of association to the plant: the endosphere, the rhizosphere and the root surrounding soil that were compared to the uncultivated soil. Bacterial community structure and diversity, determined by using Denaturing Gradient Gel Electrophoresis, differed according to the microhabitat, indicating a selective pressure determined by the plant activity. Similarly, culturable bacteria genera showed different distribution in the three root system fractions. <em>Bacillus</em> spp. (68% of the isolates) were mainly recovered from the endosphere, while rhizosphere and the root surrounding soil fractions were dominated by <em>Klebsiella</em> spp. (61% and 44% respectively). Most of the isolates (95%) presented <em>in vitro</em> multiple plant growth promoting (PGP) activities and stress resistance capabilities, but their distribution was different among the root system fractions analyzed, with enhanced abilities for <em>Bacillus</em> and the rhizobacteria strains. We show that the <em>C. annuum</em> rhizosphere under desert farming enriched populations of PGP bacteria capable of enhancing plant photosynthetic activity and biomass synthesis (up to 40%) under drought stress.</p> <h3>Conclusions/Significance</h3><p>Crop cultivation provides critical ecosystem services in arid lands with the plant root system acting as a “resource island” able to attract and select microbial communities endowed with multiple PGP traits that sustain plant development under water limiting conditions.</p> </div