Liposomes as selenium nanocarriers for foliar application to wheat plants: A biofortification strategy.

In the present work, liposomes have been used as nanocarriers in the biofortification of wheat plants with selenium (Se) through foliar application. Liposomal formulations were prepared using 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and Phospholipon®90H (P90H) (average size <100 nm), loaded with different concentrations of inorganic Se (selenite and selenate) and applied twice to the plants in the stage of vegetative growth. Liposomes enhanced Se uptake by wheat plants compared to direct application. The highest Se enrichment was achieved using the phospholipid DPPC and a concentration of 1000 µmol·L-1 of Se without affecting the biomass, chlorophylls, carotenoids, and the concentration of mineral nutrients of the plants. The chemical speciation of Se in the plants was further investigated by X-ray absorption spectroscopy (XAS). The results from XAS spectra revealed that most of the inorganic Se was transformed to organic Se and that the use of liposomes influenced the proportion of C-Se-C over C-Se-Se-C species.

Phytochemical Profile, Bioactive Properties, and Se Speciation of Se-Biofortified Red Radish (Raphanus sativus), Green Pea (Pisum sativum), and Alfalfa (Medicago sativa) Microgreens.

The impact of selenium (Se) enrichment on bioactive compounds and sugars and Se speciation was assessed on different microgreens (green pea, red radish, and alfalfa). Sodium selenite and sodium selenate at a total concentration of 20 µM (1:1) lead to a noticeable Se biofortification (40-90 mg Se kg-1 DW). In green pea and alfalfa, Se did not negatively impact phenolics and antioxidant capacity, while in red radish, a significant decrease was found. Regarding photosynthetic parameters, Se notably increased the level of chlorophylls and carotenoids in green pea, decreased chlorophyll levels in alfalfa, and had no effect on red radish. Se treatment significantly increased sugar levels in green pea and alfalfa but not in red radish. Red radish had the highest Se amino acid content (59%), followed by alfalfa (34%) and green pea (28%). These findings suggest that Se-biofortified microgreens have the potential as functional foods to improve Se intake in humans.

Selenium biofortification of microgreens: Influence on phytochemicals, pigments and nutrients.

Kale (Brassica oleracea L. var. sabellica L.), kohlrabi (Brassica oleracea L. var. gongylodes L.) and wheat (Triticum aestivum L. cv. Bancal) microgreens were cultivated in presence of selenium 20 µmol L-1 as sodium selenite and sodium selenate mixture. The influence of this biofortification process was evaluated in terms of biomass production, total Se, macro- and micronutrients concentration, polyphenols, antioxidant activity, chlorophylls and carotenoids levels and total soluble proteins content. The results obtained have shown a significant concentration of total Se in the biofortified microgreens of kale (133 µg Se·g-1 DW) and kohlrabi (127 µg Se·g-1 DW) higher than that obtained for wheat (28 µg Se·g-1 DW). The Se uptake in all the species did not produce oxidative damage to the plants reflected in the bioactive compounds, antioxidant capacity or pigments concentration. These Se-enriched microgreens may contribute to the recommended intake of this nutrient in human diet as to overcome Se-deficiency.

Mercury speciation in selenium enriched wheat plants hydroponically exposed to mercury pollution.

Mercury (Hg) pollution in agricultural soils and its potential pathway to the human food chain can pose a serious health concern. Understanding the pathway of Hg in plants and how the speciation may change upon interaction with other elements used for biofortification can be critical to assess the real implications for the final plant-based product. In that respect, selenium (Se) biofortification of crops grown in Se-poor soil regions is becoming a common practice to overcome Se deficient diets. Therefore, it is important to assess the interplay between these two elements since Se may form complexes with Hg reducing its bioavailability and toxicity. In this work, the speciation of Hg in wheat plants grown hydroponically under the presence of Hg (HgCl2) and biofortified with Se (selenite, selenate, or a 1:1 mixture of both) has been investigated by X-ray absorption spectroscopy at the Hg L3-edge. The main Hg species found in wheat grains was the highly toxic methylmercury. It was found that the Se-biofortification of wheat did not prevent, in general, the Hg translocation to grains. Only the 1:1 mixture treatment seemed to have an effect in reducing the levels of Hg and the presence of methylmercury in grains.

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.

Direct and indirect selenium speciation in biofortified wheat: A tale of two techniques.

Wheat can be biofortified with different inorganic selenium (Se) forms, selenite or selenate. The choice of Se source influences the physiological response of the plant and the Se metabolites produced. We looked at selenium uptake, distribution and metabolization in wheat exposed to selenite, selenate and a 1:1 molar mixture of both to determine the impact of each treatment on the Se speciation in roots, shoots, and grains. To achieve a comprehensive quantification of the Se species, the complementarity of high-performance liquid chromatography coupled with inductively coupled plasma mass spectrometry and X-ray absorption spectroscopy was exploited. This approach allowed the identification of the six main selenium species: selenomethionine, selenocysteine, selenocystine, selenite, selenate, and elemental selenium. The three treatments resulted in similar total selenium concentration in grains, 90-150 mg Se kg-1 , but produced different effects in the plant. Selenite enhanced root accumulation (66% of selenium) and induced the maximum toxicity, whereas selenate favored shoot translocation (46%). With the 1:1 mixture, selenium was distributed along the plant generating lower toxicity. Although all conditions resulted in >92% of organic selenium in the grain, selenate produced mainly C-Se-C forms, such as selenomethionine, while selenite (alone or in the mixture) enhanced the production of C-Se-Se-C forms, such as selenocystine, modifying the selenoamino acid composition. These results provide a better understanding of the metabolization of selenium species which is key to minimize plant toxicity and any concomitant effect that may arise due to Se-biofortification.

Grapevine response to a Dittrichia viscosa extract and a Bacillus velezensis strain.

The present study aims to evaluate the response of the three Mediterranean local grapevines 'Garnacha Blanca', 'Garnacha Tinta', and 'Macabeo' to treatments with biocontrol products, namely a botanical extract (Akivi, Dittrichia viscosa extract) and a beneficial microorganism (Bacillus UdG, Bacillus velezensis). A combination of transcriptomics and metabolomics approaches were chosen in order to study grapevine gene expression and to identify gene marker candidates, as well as, to determine differentially concentrated grapevine metabolites in response to biocontrol product treatments. Grapevine plants were cultivated in greenhouse under controlled conditions and submitted to the treatments. Thereafter, leaves were sampled 24h after treatment to carry out the gene expression study by RT-qPCR for the three cultivars and by RNA-sequencing for 'Garnacha Blanca'. Differentially expressed genes (DEGs) were investigated for both treatments and highly influenced DEGs were selected to be tested in the three cultivars as treatment gene markers. In addition, the extraction of leaf components was performed to quantify metabolites, such as phytohormones, organic acids, and phenols. Considering the upregulated and downregulated genes and the enhanced metabolites concentrations, the treatments had an effect on jasmonic acid, ethylene, and phenylpropanoids defense pathways. In addition, several DEG markers were identified presenting a stable overexpression after the treatments in the three grapevine cultivars. These gene markers could be used to monitor the activity of the products in field treatments. Further research will be necessary to confirm these primary results under field conditions.

Identifying the Specific Root Microbiome of the Hyperaccumulator Noccaea brachypetala Growing in Non-metalliferous Soils.

Noccaea brachypetala is a close relative of Noccaea caerulescens, a model plant species used in metal hyperaccumulation studies. In a previous survey in the Catalan Pyrenees, we found two occidental and two oriental N. brachypetala populations growing on non-metalliferous soils, with accumulated high concentrations of Cd and Zn. Our hypothesis was that the microbiome companion of the plant roots may influence the ability of these plants to absorb metals. We performed high-throughput sequencing of the bacterial and fungal communities in the rhizosphere soil and rhizoplane fractions. The rhizobiomes and shoot ionomes of N. brachypetala plants were analyzed along with those from other non-hyperaccumulator Brassicaceae species found at the same sampling locations. The analyses revealed that microbiome richness and relative abundance tended to increase in N. brachypetala plants compared to non-hyperaccumulator species, regardless of plant location. We confirmed that the root compartment is a key factor in describing the community composition linked to the cohabiting Brassicaceae species, and the rhizoplane fraction contained the specific and rare taxa associated with each species. N. brachypetala plants harbored a similar relative abundance of fungi compared to the other plant hosts, but there was a notable reduction in some specific taxa. Additionally, we observed an enrichment in the hyperaccumulator rhizoplane of previously described metal-tolerant bacteria and bacteria involved in nitrogen cycling. The bacteria involved in the nitrogen cycle could contribute indirectly to the hyperaccumulator phenotype by improving soil quality and fertility. Our results indicate that N. brachypetala captures a particular prokaryotic community from the soil. This particular prokaryotic community may benefit the extraction of metal ions and/or improve plant nutrition. Our research identified satellite groups associated with the root niche of a hyperaccumulator plant that may assist in improving biological strategies in heavy metal remediation.

Co-application of Se and a biostimulant at different wheat growth stages: Influence on grain development.

An appropriate selenium intake can be beneficial for human health. Se-biofortified food in Se-deficient regions is becoming an increasingly common practice but there are still issues to be addressed regarding the observed Se-induced toxicity to the plant. In this respect, plant biostimulants are used to enhance nutrition efficiency, abiotic stress tolerance and crop quality. In this work, the efficacy of a plant biostimulant to counteract the Se-induced stress in wheat plants is experimentally assessed. The co-application of different Se-biofortification treatments and the biostimulant at different growth stages (tillering or heading stage) was investigated. The use of micro focused X-ray spectroscopy allows us to confirm organic Se species to be the main Se species found in wheat grain and that the proportion of organic Se species is only slightly affected by the Se application stage. Our study proves that the biostimulant had a key role in the enhancement of both the amount of grains produced per spike and their dry biomass without hindering Se enrichment process, neither diminishing the Se concentration nor massively disrupting the Se species present. This information will be useful to minimize both plant toxicity and economic cost towards a more effective and plant healthy selenium supplementation.

A Role for Zinc in Plant Defense Against Pathogens and Herbivores.

Pests and diseases pose a threat to food security, which is nowadays aggravated by climate change and globalization. In this context, agricultural policies demand innovative approaches to more effectively manage resources and overcome the ecological issues raised by intensive farming. Optimization of plant mineral nutrition is a sustainable approach to ameliorate crop health and yield. Zinc is a micronutrient essential for all living organisms with a key role in growth, development, and defense. Competition for Zn affects the outcome of the host-attacker interaction in both plant and animal systems. In this review, we provide a clear framework of the different strategies involving low and high Zn concentrations launched by plants to fight their enemies. After briefly introducing the most relevant macro- and micronutrients for plant defense, the functions of Zn in plant protection are summarized with special emphasis on superoxide dismutases (SODs) and zinc finger proteins. Following, we cover recent meaningful studies identifying Zn-related passive and active mechanisms for plant protection. Finally, Zn-based strategies evolved by pathogens and pests to counteract plant defenses are discussed.

Snails prefer it sweet: A multifactorial test of the metal defence hypothesis.

Metal defence against insect herbivory in hyperaccumulator plants is well documented. However, there are contradictory results regarding protection against snails. According to the joint effects hypothesis, inorganic and organic defences cooperate in plant protection. To test this hypothesis, we explored the relationships between snail (Cantareus aspersus) feeding and multiple inorganic and organic leaf components in the Cd hyperaccumulator plant Noccaea praecox. Plants grouped by rosette size growing in nutrient solution supplemented or not with 50 µM Cd were offered to the snails. After 3 days of snail feeding, the plants and snails were analysed. In addition to Cd concentrations, we analysed leaves for nutritional factors (sugar and protein), defence-related compounds (glucosinolates, phenolics, tannins, salicylic acid and jasmonate) and essential mineral nutrients. Cadmium concentrations in the snails and in snail excrements were also analysed. Snails preferentially fed on plants grown without Cd. Medium-sized plants exposed to Cd were the least consumed. Snail excrements from this trial weighed less and had higher Cd concentrations than those from other treatments. Cadmium increased salicylate and jasmonate production. A positive relationship between jasmonate levels and the number of attacked leaves was found. Principal component analysis revealed that leaf sugar concentration was the main factor positively affecting snails' leaf consumption, while leaf Cd had a negative but weaker influence. In conclusion, leaf sugar concentration mainly governs snails' feeding preferences. High leaf Cd concentrations do not deter herbivores from attacking leaves, but they do reduce leaf consumption. Our results clearly support the joint effects hypothesis.

Zinc triggers signaling mechanisms and defense responses promoting resistance to Alternaria brassicicola in Arabidopsis thaliana.

According to the elemental defense hypothesis the accumulation of trace elements by plants may substitute for organic defenses, while the joint effects hypothesis proposes that trace elements and organic defenses can have additive or synergistic effects against pathogens or herbivores. To evaluate these hypotheses the response of the pathosystem Alternaria brassicicola-Arabidopsis thaliana to control (2µM) and surplus (12µM) Zn was evaluated using the camalexin deficient mutant pad3-1 and mtp1-1, a mutant with impaired Zn vacuolar storage, along with the corresponding wildtypes. In vitro, a 50% inhibition of fungal growth was achieved by 440µM Zn. A. thaliana leaves could accumulate equivalent concentrations without harm. In fact, surplus Zn enhanced the resistance of A. thaliana to fungal attack in Columbia (Col-0), Wassilewskija (WS), and mtp1-1. However, surplus Zn was unable to protect pad3-1 demonstrating that Zn cannot substitute for camalexin, the main organic defense in A. thaliana. High, non phytotoxic leaf Zn concentrations enhanced the resistance to A. brassicicola of A. thaliana genotypes able to produce camalexin. This was mainly due to Zn-induced enhancement of the JA/ETH signaling pathway leading to enhanced PAD3 expression. These results support the joint effects hypothesis and highlight the importance of adequate Zn supply for reinforced pathogen resistance.

High salinity helps the halophyte Sesuvium portulacastrum in defense against Cd toxicity by maintaining redox balance and photosynthesis.

MAIN CONCLUSION: NaCl alleviates Cd toxicity in Sesvium portulacastrum by maintaining plant water status and redox balance, protecting chloroplasts structure and inducing some potential Cd (2+) chelators as GSH and proline. It has been demonstrated that NaCl alleviates Cd-induced growth inhibition in the halophyte Sesuvium portulacastrum. However, the processes that mediate this effect are still unclear. In this work we combined physiological, biochemical and ultrastructural studies to highlight the effects of salt on the redox balance and photosynthesis in Cd-stressed plants. Seedlings were exposed to different Cd concentrations (0, 25 and 50 µM Cd) combined with low (0.09 mM) (LS), or high (200 mM) NaCl (HS) in hydroponic culture. Plant-water relations, photosynthesis rate, leaf gas exchange, chlorophyll fluorescence, chloroplast ultrastructure, and proline and glutathione concentrations were analyzed after 1 month of treatment. In addition, the endogenous levels of stress-related hormones were determined in plants subjected to 25 µM Cd combined with both NaCl concentrations. In plants with low salt supply (LS), Cd reduced growth, induced plant dehydration, disrupted chloroplast structure and functioning, decreased net CO2 assimilation rate (A) and transpiration rate (E), inhibited the maximum potential quantum efficiency (Fv/Fm) and the quantum yield efficiency (Φ PSII) of PSII, and enhanced the non-photochemical quenching (NPQ). The addition of 200 mM NaCl (HS) to the Cd-containing medium culture significantly mitigated Cd phytotoxicity. Hence, even at similar internal Cd concentrations, HS-Cd plants were less affected by Cd than LS-Cd ones. Hence, 200 mM NaCl significantly alleviates Cd-induced toxicity symptoms, growth inhibition, and photosynthesis disturbances. The cell ultrastructure was better preserved in HS-Cd plants but affected in LS-Cd plants. The HS-Cd plants showed also higher concentrations of reduced glutathione (GSH), proline and jasmonic acid (JA) than the LS-Cd plants. However, under LS-Cd conditions, plants maintained higher concentration of salicylic acid (SA) and abscisic acid (ABA) than the HS-Cd ones. We conclude that in S. portulacastrum alleviation of Cd toxicity by NaCl is related to the modification of GSH and proline contents as well as stress hormone levels thus protecting redox balance and photosynthesis.

Differential activation of genes related to aluminium tolerance in two contrasting rice cultivars.

Rice (Oryza sativa) is a highly Al-tolerant crop. Among other mechanisms, a higher expression of STAR1/STAR2 (sensitive to Al rhizotoxicity1/2) genes and of Nrat1 (NRAMP Aluminium Transporter 1), and ALS1 (Aluminium sensitive 1) can at least in part be responsible for the inducible Al tolerance in this species. Here we analysed the responses to Al in two contrasting rice varieties. All analysed toxicity/tolerance markers (root elongation, Evans blue, morin and haematoxylin staining) indicated higher Al-tolerance in variety Nipponbare, than in variety Modan. Nipponbare accumulated much less Al in the roots than Modan. Aluminium supply caused stronger expression of STAR1 in Nipponbare than in Modan. A distinctively higher increase of Al-induced abscisic acid (ABA) accumulation was found in the roots of Nipponbare than in Modan. Highest ABA levels were observed in Nipponbare after 48 h exposure to Al. This ABA peak was coincident in time with the highest expression level of STAR1. It is proposed that ABA may be required for cell wall remodulation facilitated by the enhanced UDP-glucose transport to the walls through STAR1/STAR2. Contrastingly, in the roots of Modan the expression of both Nrat1 coding for a plasma membrane Al-transporter and of ALS1 coding for a tonoplast-localized Al transporter was considerably enhanced. Moreover, Modan had a higher Al-induced expression of ASR1 a gene that has been proposed to code for a reactive oxygen scavenging protein. In conclusion, the Al-exclusion strategy of Nipponbare, at least in part mediated by STAR1 and probably regulated by ABA, provided better protection against Al toxicity than the accumulation and internal detoxification strategy of Modan mediated by Nrat1, ALS1 and ARS1.

Influence of the Ca/Mg ratio on Cu resistance in three Silene armeria ecotypes adapted to calcareous soil or to different, Ni- or Cu-enriched, serpentine sites.

This hydroponic study addresses the influence of low (0.3) and high (4.0) Ca/Mg molar ratios on Cu resistance of Silene armeria ecotypes from different habitats: a calcareous soil (ecotype Cadriano), a Ni-rich serpentine site (ecotype Prinzera), and an acid Cu-mine spoil soil containing serpentinite (ecotype Vigonzano). Under control conditions, without excess Cu, only Cadriano was negatively affected by the low Ca/Mg ratio. Under both low and high Ca/Mg ratios Cu resistance followed the order Vigonzano more more than Prinzera > Cadriano. More efficient Cu exclusion accounted for enhanced Cu resistance in Prinzera. The low Ca/Mg ratio increased Cu uptake in Prinzera but did not worsen toxicity effects; i.e. the plants had higher internal Cu effect concentrations. In Vigonzano Cu resistance was enhanced by the low Ca/Mg ratio. This was due only in part to better Cu exclusion. Magnesium-induced tolerance to higher Cu tissue concentrations appears to be in ecotypes from serpentine and acid mine spoils, but not in plants from calcareous soil, the exposure to low Ca/Mg ratio favours internal detoxification of Cu by means of more efficient chelation and compartmentation.

The role of phytochelatins in constitutive and adaptive heavy metal tolerances in hyperaccumulator and non-hyperaccumulator metallophytes.

Using the gamma-glutamylcysteine synthetase inhibitor, L-buthionine-[S,R]-sulphoximine (BSO), the role for phytochelatins (PCs) was evaluated in Cu, Cd, Zn, As, Ni, and Co tolerance in non-metallicolous and metallicolous, hypertolerant populations of Silene vulgaris (Moench) Garcke, Thlaspi caerulescens J.&C. Presl., Holcus lanatus L., and Agrostis castellana Boiss. et Reuter. Based on plant-internal PC-thiol to metal molar ratios, the metals' tendency to induce PC accumulation decreased in the order As/Cd/Cu > Zn > Ni/Co, and was consistently higher in non-metallicolous plants than in hypertolerant ones, except for the case of As. The sensitivities to Cu, Zn, Ni, and Co were consistently unaffected by BSO treatment, both in non-metallicolous and hypertolerant plants, suggesting that PC-based sequestration is not essential for constitutive tolerance or hypertolerance to these metals. Cd sensitivity was considerably increased by BSO, though exclusively in plants lacking Cd hypertolerance, suggesting that adaptive cadmium hypertolerance is not dependent on PC-mediated sequestration. BSO dramatically increased As sensitivity, both in non-adapted and As-hypertolerant plants, showing that PC-based sequestration is essential for both normal constitutive tolerance and adaptive hypertolerance to this metalloid. The primary function of PC synthase in plants and algae remains elusive.