Molecular insights into the mutualism that induces iron deficiency tolerance in sorghum inoculated with Trichoderma harzianum.

Iron (Fe) deficiency is a common mineral stress in plants, including sorghum. Although the soil fungus Trichoderma harzianum has been shown to mitigate Fe deficiency in some circumstances, neither the range nor mechanism(s) of this process are well understood. In this study, high pH-induced Fe deficiency in sorghum cultivated in pots with natural field soil exhibited a significant decrease in biomass, photosynthetic rate, transpiration rate, stomatal conductance, water use efficiency, and Fe-uptake in both the root and shoot. However, the establishment of T. harzianum colonization in roots of Fe-deprived sorghum showed significant improvements in morpho-physiological traits, Fe levels, and redox status. Molecular detection of the fungal ThAOX1 (L-aminoacid oxidase) gene showed the highest colonization of T. harzianum in the root tips of Fe-deficient sorghum, a location thus targeted for further analysis. Expression studies by RNA-seq and qPCR in sorghum root tips revealed a significant upregulation of several genes associated with Fe uptake (SbTOM2), auxin synthesis (SbSAURX15), nicotianamine synthase 3 (SbNAS3), and a phytosiderophore transporter (SbYS1). Also induced was the siderophore synthesis gene (ThSIT1) in T. harzianum, a result supported by biochemical evidence for elevated siderophore and IAA (indole acetic acid) levels in roots. Given the high affinity of fungal siderophore to chelate insoluble Fe3+ ions, it is likely that elevated siderophore released by T. harzianum led to Fe(III)-siderophore complexes in the rhizosphere that were then transported into roots by the induced SbYS1 (yellow-stripe 1) transporter. In addition, the observed induction of several plant peroxidase genes and ABA (abscisic acid) under Fe deficiency after inoculation with T. harzianum may have helped induce tolerance to Fe-deficiency-induced oxidative stress and adaptive responses. This is the first mechanistic explanation for T. harzianum's role in helping alleviate Fe deficiency in sorghum and suggests that biofertilizers using T. harzianum will improve Fe availability to crops in high pH environments.

Regulation of Phytosiderophore Release and Antioxidant Defense in Roots Driven by Shoot-Based Auxin Signaling Confers Tolerance to Excess Iron in Wheat.

Iron (Fe) is essential but harmful for plants at toxic level. However, how wheat plants tolerate excess Fe remains vague. This study aims at elucidating the mechanisms underlying tolerance to excess Fe in wheat. Higher Fe concentration caused morpho-physiological retardation in BR 26 (sensitive) but not in BR 27 (tolerant). Phytosiderophore and 2-deoxymugineic acid showed no changes in BR 27 but significantly increased in BR 26 due to excess Fe. Further, expression of TaSAMS. TaDMAS1, and TaYSL15 significantly downregulated in BR 27 roots, while these were upregulated in BR 26 under excess Fe. It confirms that inhibition of phytosiderophore directs less Fe accumulation in BR 27. However, phytochelatin and expression of TaPCS1 and TaMT1 showed no significant induction in response to excess Fe. Furthermore, excess Fe showed increased catalase, peroxidase, and glutathione reductase activities along with glutathione, cysteine, and proline accumulation in roots in BR 27. Interestingly, BR 27 self-grafts and plants having BR 26 rootstock attached to BR 27 scion had no Fe-toxicity induced adverse effect on morphology but showed BR 27 type expressions, confirming that shoot-derived signal triggering Fe-toxicity tolerance in roots. Finally, auxin inhibitor applied with higher Fe concentration caused a significant decline in morpho-physiological parameters along with increased TaSAMS and TaDMAS1 expression in roots of BR 27, revealing the involvement of auxin signaling in response to excess Fe. These findings propose that tolerance to excess Fe in wheat is attributed to the regulation of phytosiderophore limiting Fe acquisition along with increased antioxidant defense in roots driven by shoot-derived auxin signaling.

Role of Silicon Counteracting Cadmium Toxicity in Alfalfa (Medicago sativa L.).

Cadmium (Cd) is one of the most phytotoxic elements causing an agricultural problem and human health hazards. This work investigates whether and how silicon (Si) ameliorates Cd toxicity in Alfalfa. The addition of Si in Cd-stressed plants caused significant improvement in morpho-physiological features as well as total protein and membrane stability, indicating that Si does have critical roles in Cd detoxification in Alfalfa. Furthermore, Si supplementation in Cd-stressed plants showed a significant decrease in Cd and Fe concentrations in both roots and shoots compared with Cd-stressed plants, revealing that Si-mediated tolerance to Cd stress is associated with Cd inhibition in Alfalfa. Results also showed no significant changes in the expression of two metal chelators [MsPCS1 (phytochelatin synthase) and MsMT2 (metallothionein)] and PC (phytochelatin) accumulation, indicating that there may be no metal sequestration or change in metal sequestration following Si application under Cd stress in Alfalfa. We further performed a targeted study on the effect of Si on Fe uptake mechanisms. We observed the consistent reduction in Fe reductase activity, expression of Fe-related genes [MsIRT1 (Fe transporter), MsNramp1 (metal transporter) and OsFRO1 (ferric chelate reductase] and Fe chelators (citrate and malate) by Si application to Cd stress in roots of Alfalfa. These results support that limiting Fe uptake through the down-regulation of Fe acquisition mechanisms confers Si-mediated alleviation of Cd toxicity in Alfalfa. Finally, an increase of catalase, ascorbate peroxidase, and superoxide dismutase activities along with elevated methionine and proline subjected to Si application might play roles, at least in part, to reduce H2O2 and to provide antioxidant defense against Cd stress in Alfalfa. The study shows evidence of the effect of Si on alleviating Cd toxicity in Alfalfa and can be further extended for phytoremediation of Cd toxicity in plants.

Silicate reduces cadmium uptake into cells of wheat.

Cadmium (Cd) is a health threat all over the world and high Cd content in wheat causes high Cd intake. Silicon (Si) decreases cadmium content in wheat grains and shoot. This work investigates whether and how silicate (Si) influences cadmium (Cd) uptake at the cellular level in wheat. Wheat seedlings were grown in the presence or absence of Si with or without Cd. Cadmium, Si, and iron (Fe) accumulation in roots and shoots was analysed. Leaf protoplasts from plants grown without Cd were investigated for Cd uptake in the presence or absence of Si using the fluorescent dye, Leadmium Green AM. Roots and shoots of plants subjected to all four treatments were investigated regarding the expression of genes involved in the Cd uptake across the plasma membrane (i.e. LCT1) and efflux of Cd into apoplasm or vacuole from the cytosol (i.e. HMA2). In addition, phytochelatin (PC) content and PC gene (PCS1) expression were analysed. Expression of iron and metal transporter genes (IRT1 and NRAMP1) were also analysed. Results indicated that Si reduced Cd accumulation in plants, especially in shoot. Si reduced Cd transport into the cytoplasm when Si was added both directly during the uptake measurements and to the growth medium. Silicate downregulated LCT1 and HMA2 and upregulated PCS1. In addition, Si enhanced PC formation when Cd was present. The IRT1 gene, which was downregulated by Cd was upregulated by Si in root and shoot facilitating Fe transport in wheat. NRAMP1 was similarly expressed, though the effect was limited to roots. This work is the first to show how Si influences Cd uptake on the cellular level.

Mechanisms associated with Fe-deficiency tolerance and signaling in shoots of Pisum sativum.

Mechanisms of Fe-deficiency tolerance and signaling were investigated in shoots of Santi (deficiency tolerant) and Parafield (deficiency intolerant) pea genotypes using metabolomic and physiological approaches. From metabolomic studies, Fe deficiency induced significant increases in N-, S- and tricarboxylic acid cycle metabolites in Santi but not in Parafield. Elevated N metabolites reflect an increase in N-recycling processes. Increased glutathione and S-metabolites suggest better protection of pea plants from Fe-deficiency-induced oxidative stress. Furthermore, Fe-deficiency induced increases in citrate and malate in leaves of Santi suggests long-distance transport of Fe is promoted by better xylem unloading. Supporting a role of citrate in the deficiency tolerance mechanism, physiological experiments showed higher Fe and citrate in the xylem of Santi. Reciprocal-grafting experiments confirm that the Fe-deficiency signal driving root Fe reductase and proton extrusion activity is generated in the shoot. Finally, our studies show that auxin can induce increased Fe-reductase activity and proton extrusion in roots. This article identifies several mechanisms in shoots associated with the differential Fe-deficiency tolerance of genotypes within a species, and provides essential background for future efforts to improve the Fe content and deficiency tolerance in peas.

Natural variation for Fe-efficiency is associated with upregulation of Strategy I mechanisms and enhanced citrate and ethylene synthesis in Pisum sativum L.

Iron (Fe)-deficiency is a common abiotic stress in Pisum sativum L. grown in many parts of the world. The aim of the study was to investigate variation in tolerance to Fe deficiency in two pea genotypes, Santi (Fe-efficient) and Parafield (Fe-inefficient). Fe deficiency caused greater declines in chlorophyll score, leaf Fe concentration and root-shoot development in Parafield compared to Santi, suggesting greater Fe-efficiency in Santi. Fe chelate reductase activity and ethylene production were increased in the roots of Santi and to a lesser extent in Parafield under Fe deficiency, while proton extrusion was only occurred in Santi. Moreover, expression of the Fe chelate reductase gene, FRO1, and Fe transporter, RIT1 were upregulated in Fe-deficient roots of Santi. Expression of HA1 (proton extrusion) was also significantly higher in Santi when compared to Parafield grown in Fe-deficient conditions. Furthermore, the application of the ethylene biosynthesis inhibitor, 1-aminoisobutyric acid reduced the Fe chelate reductase activity, supporting a direct role for ethylene in its induction. A significant increase in root citrate was only observed in Santi under Fe deficiency indicating a role for citrate in the Fe-efficiency mechanism. Taken together, our physiological and molecular data indicate that genotypic variation in tolerance to Fe deficiency in Santi and Parafield plants is a result of variation in a number of Strategy I mechanisms and also suggest a direct role for ethylene in Fe reductase activity. The pea cultivar, Santi provides a new source of Fe-efficiency that can be exploited to breed more Fe-efficient peas.