Nutritional Composition of Post-Catastrophic Foods.

In addition to current challenges in food production arising from climate change, soil salinization, drought, flooding, and human-caused disruption, abrupt sunlight reduction scenarios (ASRS), e.g., a nuclear winter, supervolcano eruption, or large asteroid or comet strike, are catastrophes that would severely disrupt the global food supply and decimate normal agricultural practices. In such global catastrophes, teragrams of particulate matter, such as aerosols of soot, dust, and sulfates, would be injected into the stratosphere and block sunlight for multiple years. The reduction of incident sunlight would cause a decrease in temperature and precipitation and major shifts to climate patterns leading to devastating reductions in agricultural production of traditional food crops. To survive a catastrophic ASRS or endure current and future disasters and famines, humans might need to rely on post-catastrophic foods, or those that could be foraged, grown, or produced under the new climate conditions to supplement reduced availability of traditional foods. These foods have sometimes been referred to as emergency, alternate, or resilient foods in the literature. While there is a growing body of work that summarizes potential post-catastrophic foods and their nutritional profiles based on existing data in the literature, this article documents a list of protocols to experimentally determine fundamental nutritional properties of post-catastrophic foods that can be used to assess the relative contributions of those foods to a balanced human diet that meets established nutritional requirements while avoiding toxic levels of nutrients. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Total digestible glucans Basic Protocol 2: Apparent protein digestibility Basic Protocol 3: Vitamins B1, B3, B9, C, and D2 by HPLC Basic Protocol 4: Total antioxidant activity (DPPH-scavenging activity) Basic Protocol 5: Total phenolic compounds (Folin-Ciocalteu reagent method) Basic Protocol 6: Mineral content by ICP-OES.

Zinc biofortification through seed nutri-priming using alternative zinc sources and concentration levels in pea and sunflower microgreens.

Micronutrient deficiencies caused by malnutrition and hidden hunger are a growing concern worldwide, exacerbated by climate change, COVID-19, and conflicts. A potentially sustainable way to mitigate such challenges is the production of nutrient-dense crops through agronomic biofortification techniques. Among several potential target crops, microgreens are considered suitable for mineral biofortification because of their short growth cycle, high content of nutrients, and low level of anti-nutritional factors. A study was conducted to evaluate the potential of zinc (Zn) biofortification of pea and sunflower microgreens via seed nutri-priming, examining the effect of different Zn sources (Zn sulfate, Zn-EDTA, and Zn oxide nanoparticles) and concentrations (0, 25, 50, 100, and 200 ppm) on microgreen yield components; mineral content; phytochemical constituents such as total chlorophyll, carotenoids, flavonoids, anthocyanin, and total phenolic compounds; antioxidant activity; and antinutrient factors like phytic acid. Treatments were arranged in a completely randomized factorial block design with three replications. Seed soaked in a 200 ppm ZnSO4 solution resulted in higher Zn accumulation in both peas (126.1%) and sunflower microgreens (229.8%). However, an antagonistic effect on the accumulation of other micronutrients (Fe, Mn, and Cu) was seen only in pea microgreens. Even at high concentrations, seed soaking in Zn-EDTA did not effectively accumulate Zn in both microgreens' species. ZnO increased the chlorophyll, total phenols, and antioxidant activities compared to Zn-EDTA. Seed soaking in ZnSO4 and ZnO solutions at higher concentrations resulted in a lower phytic acid/Zn molar ratio, suggesting the higher bioaccessibility of the biofortified Zn in both pea and sunflower microgreens. These results suggest that seed nutrient priming is feasible for enriching pea and sunflower microgreens with Zn. The most effective Zn source was ZnSO4, followed by ZnO. The optimal concentration of Zn fertilizer solution should be selected based on fertilizer source, target species, and desired Zn-enrichment level.

Alteration of iron responsive gene expression in Arabidopsis glutaredoxin S17 loss of function plants with or without iron stress.

Iron (Fe) is a mineral nutrient and a metal cofactor essential for plants. Iron limitation can have detrimental effects on plant growth and development, while excess iron inside plant cells leads to oxidative damage. As a result, plants have evolved complex regulatory networks to respond to fluctuations in cellular iron concentrations. The mechanisms that regulate these responses however, are not fully understood. Heterologous expression of an Arabidopsis thaliana monothiol glutaredoxin S17 (GRXS17) suppresses the over-accumulation of iron in the Saccharomyces cerevisiae Grx3/Grx4 mutant and disruption of GRXS17 causes plant sensitivity to exogenous oxidants and iron deficiency stress. GRXS17 may act as an important regulator in the plant's ability to respond to iron deficiency stress and maintain redox homeostasis. Here, we extend this investigation by analyzing iron-responsive gene expression of the Fer-like iron deficiency-induced transcription factor (FIT) network (FIT, IRT1, FRO1, and FRO2) and the bHLH transcription factor POPEYE (PYE) network (PYE, ZIF1, FRO3, NAS4, and BTS) in GRXS17 KO plants and wildtype controls grown under iron sufficiency and deficiency conditions. Our findings suggest that GRXS17 is required for tolerance to iron deficiency, and plays a negative regulatory role under conditions of iron sufficiency.

Species-Specific Duplication Event Associated with Elevated Levels of Nonstructural Carbohydrates in Sorghum bicolor.

Simple sugars are the essential foundation to plant life, and thus, their production, utilization, and storage are highly regulated processes with many complex genetic controls. Despite their importance, many of the genetic and biochemical mechanisms remain unknown or uncharacterized. Sorghum, a highly productive, diverse C4 grass important for both industrial and subsistence agricultural systems, has considerable phenotypic diversity in the accumulation of nonstructural sugars in the stem. We use this crop species to examine the genetic controls of high levels of sugar accumulation, identify genetic mechanisms for the accumulation of nonstructural sugars, and link carbon allocation with iron transport. We identify a species-specific tandem duplication event controlling sugar accumulation using genome-wide association analysis, characterize multiple allelic variants causing increased sugar content, and provide further evidence of a putative neofunctionalization event conferring adaptability in Sorghum bicolor Comparative genomics indicate that this event is unique to sorghum which may further elucidate evolutionary mechanisms for adaptation and divergence within the Poaceae. Furthermore, the identification and characterization of this event was only possible with the continued advancement and improvement of the reference genome. The characterization of this region and the process in which it was discovered serve as a reminder that any reference genome is imperfect and is in need of continual improvement.

Mitochondrial Iron Transporters (MIT1 and MIT2) Are Essential for Iron Homeostasis and Embryogenesis in Arabidopsis thaliana.

Iron (Fe) is an essential nutrient for virtually all organisms, where it functions in critical electron transfer processes, like those involved in respiration. Photosynthetic organisms have special requirements for Fe due to its importance in photosynthesis. While the importance of Fe for mitochondria- and chloroplast-localized processes is clear, our understanding of the molecular mechanisms that underlie the trafficking of Fe to these compartments is not complete. Here, we describe the Arabidopsis mitochondrial iron transporters, MIT1 and MIT2, that belong to the mitochondrial carrier family (MCF) of transport proteins. MIT1 and MIT2 display considerable homology with known mitochondrial Fe transporters of other organisms. Expression of MIT1 or MIT2 rescues the phenotype of the yeast mrs3mrs4 mutant, which is defective in mitochondrial iron transport. Although the Arabidopsis mit1 and mit2 single mutants do not show any significant visible phenotypes, the double mutant mit1mit2 displays embryo lethality. Analysis of a mit1 -- /mit2 + - line revealed that MIT1 and MIT2 are essential for iron acquisition by mitochondria and proper mitochondrial function. In addition, loss of MIT function results in mislocalization of Fe, which in turn causes upregulation of the root high affinity Fe uptake pathway. Thus, MIT1 and MIT2 are required for the maintenance of both mitochondrial and whole plant Fe homeostasis, which, in turn, is important for the proper growth and development of the plant.

Directing iron transport in dicots: regulation of iron acquisition and translocation.

Iron is essential for plant growth and development, but excess iron is cytotoxic. While iron is abundant in soil, it is often a limiting nutrient for plant growth. Consequentially, plants have evolved mechanisms to tightly regulate iron uptake, trafficking and storage. Recent work has contributed to a more comprehensive picture of iron uptake, further elucidating molecular and physiological processes that aid in solubilization of iron and modulation of the root system architecture in response to iron availability. Recent progress in understanding the regulators of the iron deficiency response and iron translocation from root to shoots, and especially to seeds are noteworthy. The molecular bases of iron sensing and signaling are gradually emerging, as well.

Arabidopsis Glutaredoxin S17 Contributes to Vegetative Growth, Mineral Accumulation, and Redox Balance during Iron Deficiency.

Iron (Fe) is an essential mineral nutrient and a metal cofactor required for many proteins and enzymes involved in the processes of DNA synthesis, respiration, and photosynthesis. Iron limitation can have detrimental effects on plant growth and development. Such effects are mediated, at least in part, through the generation of reactive oxygen species (ROS). Thus, plants have evolved a complex regulatory network to respond to conditions of iron limitations. However, the mechanisms that couple iron deficiency and oxidative stress responses are not fully understood. Here, we report the discovery that an Arabidopsis thaliana monothiol glutaredoxin S17 (AtGRXS17) plays a critical role in the plants ability to respond to iron deficiency stress and maintain redox homeostasis. In a yeast expression assay, AtGRXS17 was able to suppress the iron accumulation in yeast ScGrx3/ScGrx4 mutant cells. Genetic analysis indicated that plants with reduced AtGRXS17 expression were hypersensitive to iron deficiency and showed increased iron concentrations in mature seeds. Disruption of AtGRXS17 caused plant sensitivity to exogenous oxidants and increased ROS production under iron deficiency. Addition of reduced glutathione rescued the growth and alleviates the sensitivity of atgrxs17 mutants to iron deficiency. These findings suggest AtGRXS17 helps integrate redox homeostasis and iron deficiency responses.

Integration of Experiments across Diverse Environments Identifies the Genetic Determinants of Variation in Sorghum bicolor Seed Element Composition.

Seedling establishment and seed nutritional quality require the sequestration of sufficient element nutrients. The identification of genes and alleles that modify element content in the grains of cereals, including sorghum (Sorghum bicolor), is fundamental to developing breeding and selection methods aimed at increasing bioavailable element content and improving crop growth. We have developed a high-throughput work flow for the simultaneous measurement of multiple elements in sorghum seeds. We measured seed element levels in the genotyped Sorghum Association Panel, representing all major cultivated sorghum races from diverse geographic and climatic regions, and mapped alleles contributing to seed element variation across three environments by genome-wide association. We observed significant phenotypic and genetic correlation between several elements across multiple years and diverse environments. The power of combining high-precision measurements with genome-wide association was demonstrated by implementing rank transformation and a multilocus mixed model to map alleles controlling 20 element traits, identifying 255 loci affecting the sorghum seed ionome. Sequence similarity to genes characterized in previous studies identified likely causative genes for the accumulation of zinc, manganese, nickel, calcium, and cadmium in sorghum seeds. In addition to strong candidates for these five elements, we provide a list of candidate loci for several other elements. Our approach enabled the identification of single-nucleotide polymorphisms in strong linkage disequilibrium with causative polymorphisms that can be evaluated in targeted selection strategies for plant breeding and improvement.

The diverse roles of FRO family metalloreductases in iron and copper homeostasis.

Iron and copper are essential for plants and are important for the function of a number of protein complexes involved in photosynthesis and respiration. As the molecular mechanisms that control uptake, trafficking and storage of these nutrients emerge, the importance of metalloreductase-catalyzed reactions in iron and copper metabolism has become clear. This review focuses on the ferric reductase oxidase (FRO) family of metalloreductases in plants and highlights new insights into the roles of FRO family members in metal homeostasis. Arabidopsis FRO2 was first identified as the ferric chelate reductase that reduces ferric iron-chelates at the root surface-rhizosphere interface. The resulting ferrous iron is subsequently transported across the plasma membrane of root epidermal cells by the ferrous iron transporter, IRT1. Recent work has shown that two other members of the FRO family (FRO4 and FRO5) function redundantly to reduce copper to facilitate its uptake from the soil. In addition, FROs appear to play important roles in subcellular compartmentalization of iron as FRO7 is known to contribute to delivery of iron to chloroplasts while mitochondrial family members FRO3 and FRO8 are hypothesized to influence mitochondrial metal ion homeostasis. Finally, recent studies have underscored the importance of plasma membrane-localized ferric reductase activity in leaves for photosynthetic efficiency. Taken together, these studies highlight a number of diverse roles for FROs in both iron and copper metabolism in plants.

Mitochondrial iron transport and homeostasis in plants.

Iron (Fe) is an essential nutrient for plants and although the mechanisms controlling iron uptake from the soil are relatively well understood, comparatively little is known about subcellular trafficking of iron in plant cells. Mitochondria represent a significant iron sink within cells, as iron is required for the proper functioning of respiratory chain protein complexes. Mitochondria are a site of Fe-S cluster synthesis, and possibly heme synthesis as well. Here we review recent insights into the molecular mechanisms controlling mitochondrial iron transport and homeostasis. We focus on the recent identification of a mitochondrial iron uptake transporter in rice and a possible role for metalloreductases in iron uptake by mitochondria. In addition, we highlight recent advances in mitochondrial iron homeostasis with an emphasis on the roles of frataxin and ferritin in iron trafficking and storage within mitochondria.

Transcriptome sequencing identifies SPL7-regulated copper acquisition genes FRO4/FRO5 and the copper dependence of iron homeostasis in Arabidopsis.

The transition metal copper (Cu) is essential for all living organisms but is toxic when present in excess. To identify Cu deficiency responses comprehensively, we conducted genome-wide sequencing-based transcript profiling of Arabidopsis thaliana wild-type plants and of a mutant defective in the gene encoding SQUAMOSA PROMOTER BINDING PROTEIN-LIKE7 (SPL7), which acts as a transcriptional regulator of Cu deficiency responses. In response to Cu deficiency, FERRIC REDUCTASE OXIDASE5 (FRO5) and FRO4 transcript levels increased strongly, in an SPL7-dependent manner. Biochemical assays and confocal imaging of a Cu-specific fluorophore showed that high-affinity root Cu uptake requires prior FRO5/FRO4-dependent Cu(II)-specific reduction to Cu(I) and SPL7 function. Plant iron (Fe) deficiency markers were activated in Cu-deficient media, in which reduced growth of the spl7 mutant was partially rescued by Fe supplementation. Cultivation in Cu-deficient media caused a defect in root-to-shoot Fe translocation, which was exacerbated in spl7 and associated with a lack of ferroxidase activity. This is consistent with a possible role for a multicopper oxidase in Arabidopsis Fe homeostasis, as previously described in yeast, humans, and green algae. These insights into root Cu uptake and the interaction between Cu and Fe homeostasis will advance plant nutrition, crop breeding, and biogeochemical research.

Differential expression and regulation of iron-regulated metal transporters in Arabidopsis halleri and Arabidopsis thaliana--the role in zinc tolerance.

To avoid zinc (Zn) toxicity, plants have developed a Zn homeostasis mechanism to cope with Zn excess in the surrounding soil. In this report, we uncovered the difference of a cross-homeostasis system between iron (Fe) and Zn in dealing with Zn excess in the Zn hyperaccumulator Arabidopsis halleri ssp. gemmifera and nonhyperaccumulator Arabidopsis thaliana. Arabidopsis halleri shows low expression of the Fe acquisition and deficiency response-related genes IRT1 and IRT2 compared with A. thaliana. In A. thaliana, lowering the expression of IRT1 and IRT2 through the addition of excess Fe to the medium increases Zn tolerance. Excess Zn induces significant Fe deficiency in A. thaliana and reduces Fe accumulation in shoots. By contrast, the accumulation of Fe in shoots of A. halleri was stable under various Zn treatments. Root ferric chelate reductase (FRO) activity and expression of FIT are low in A. halleri compared with A. thaliana. Overexpressing a ZIP family member IRT3 in irt1-1, rescues the Fe-deficient phenotype. A fine-tuned Fe homeostasis mechanism in A. halleri maintains optimum Fe level by Zn-regulated ZIP transporters and prevents high Zn uptake through Fe-regulated metal transporters, and in part be responsible for Zn tolerance.

Time to pump iron: iron-deficiency-signaling mechanisms of higher plants.

Iron is an essential nutrient for plants, yet it often limits plant growth. On the contrary, overaccumulation of iron within plant cells leads to oxidative stress. As a consequence, iron-uptake systems are carefully regulated to ensure that iron homeostasis is maintained. In response to iron limitation, plants induce expression of sets of activities that function at the root-soil interface to solubilize iron and subsequently transfer it across the plasma membrane of root cells. Recent advances have revealed key players in the signaling pathways that function to induce these iron-uptake responses. Transcription factors belonging to the basic helix-loop-helix, ABI3/VP1(B3), and NAC families appear to function either directly or indirectly in the upregulation of iron deficiency responses.

Chloroplast Fe(III) chelate reductase activity is essential for seedling viability under iron limiting conditions.

Photosynthesis, heme biosynthesis, and Fe-S cluster assembly all take place in the chloroplast, and all require iron. Reduction of iron via a membrane-bound Fe(III) chelate reductase is required before iron transport across membranes in a variety of systems, but to date there has been no definitive genetic proof that chloroplasts have such a reduction system. Here we report that one of the eight members of the Arabidopsis ferric reductase oxidase (FRO) family, FRO7, localizes to the chloroplast. Chloroplasts prepared from fro7 loss-of-function mutants have 75% less Fe(III) chelate reductase activity and contain 33% less iron per microgram of chlorophyll than wild-type chloroplasts. This decreased iron content is presumably responsible for the observed defects in photosynthetic electron transport. When germinated in alkaline soil, fro7 seedlings show severe chlorosis and die without setting seed unless watered with high levels of soluble iron. Overall, our results provide molecular evidence that FRO7 plays a role in chloroplast iron acquisition and is required for efficient photosynthesis in young seedlings and for survival under iron-limiting conditions.

Raising the bar for biofortification: enhanced levels of bioavailable calcium in carrots.

Recent efforts to increase the levels of Ca in the edible portions of plants have used a modified calcium/proton antiporter [known as short cation exchanger 1 (sCAX1)] to increase Ca transport into vacuoles. New work has shown that consumption of Ca-fortified carrots results in enhanced Ca absorption. These studies highlight the potential of increasing plant nutrient content through expression of a high-capacity transporter and illustrate the importance of demonstrating that the fortified nutrient is bioavailable.

The FRO2 ferric reductase is required for glycine betaine's effect on chilling tolerance in Arabidopsis roots.

FRO2 (At1g01580) codes for an NADPH-dependent ferric reductase in plasma membranes of root epidermal cells with a demonstrated role in iron uptake by plants. Ferric reductase activity has been shown to be the rate-limiting step for iron uptake in strategy I plants like Arabidopsis and in rice, but it has been unclear whether FRO genes have other physiological functions. We hypothesized that FRO2 was involved in chilling stress tolerance because its expression was upregulated by treatment of plants with glycine betaine (GB), a chemical that prevents reactive oxygen species (ROS) signaling in chilling stress. This idea was confirmed by showing that the FRO2 null mutant frd1-1 failed to respond to GB in chilling assays either in relation to root growth recovery or inhibition of ROS accumulation. Measurements of ferric reductase activity in wild-type plants treated with GB before chilling showed no significant GB effect compared with controls. In addition, 35S-FRO2 transgenics with elevated mRNA levels did not have improved chilling tolerance. However, ferric reductase activity in wild-type plants or 35S-FRO2 transgenics pretreated with GB was several-fold higher after chilling compared with non-pretreated controls. These experiments identify a new physiological function for FRO2, i.e. blocking ROS accumulation during chilling. They also suggest that GB has a major effect on FRO2 activity posttranscriptionally in the cold.

Iron-induced turnover of the Arabidopsis IRON-REGULATED TRANSPORTER1 metal transporter requires lysine residues.

Iron is an essential micronutrient but is toxic if accumulated at high levels. Thus, iron uptake and distribution in plants are controlled by precise regulatory mechanisms. IRON-REGULATED TRANSPORTER1 (IRT1) is the major high affinity iron transporter responsible for iron uptake from the soil in Arabidopsis (Arabidopsis thaliana). Previously, we showed that IRT1 is subject to posttranscriptional regulation; when expressed from the constitutive cauliflower mosaic virus 35S promoter, IRT1 protein accumulates only in iron-deficient roots. IRT1 contains an intracellular loop that may be critical for posttranslational regulation by metals. Of particular interest are a histidine (His) motif (HGHGHGH) that might bind metals and two lysine residues that could serve as attachment sites for ubiquitin. We constructed a set of mutant IRT1 alleles: IRT1H154Q, IRT1H156Q, IRT1H158Q, IRT1H160Q, IRT14HQ (quadruple His mutant), IRT1K146R, IRT1K171R, and a double mutant (IRT1K146R,K171R). Mutation of the His or lysine residues did not eliminate the ability of IRT1 to transport iron or zinc. Expression of each of the IRT1 variants and an IRT1intact construct in plants from the 35S promoter revealed that either K146 or K171 is required for iron-induced protein turnover, and 35S-IRT1K146R,K171R plants contain higher levels of iron as compared to 35S-IRT1 and wild type. Furthermore, accumulation of metals in 35S-IRT1K146R,K171R plants was not associated with an increase in ferric chelate reductase activity; this result indicates that, at least under conditions when iron is abundant, reduction of ferric iron may not be the rate-limiting step in iron uptake by strategy I plants such as Arabidopsis.

The EAR-motif of the Cys2/His2-type zinc finger protein Zat7 plays a key role in the defense response of Arabidopsis to salinity stress.

Cys2/His2-type zinc finger proteins, which contain the EAR transcriptional repressor domain, are thought to play a key role in regulating the defense response of plants to biotic and abiotic stress conditions. Although constitutive expression of several of these proteins was shown to enhance the tolerance of transgenic plants to abiotic stress, it is not clear whether the EAR-motif of these proteins is involved in this function. In addition, it is not clear whether suppression of plant growth, induced in transgenic plants by different Cys2/His2 EAR-containing proteins, is mediated by the EAR-domain. Here we report that transgenic Arabidopsis plants constitutively expressing the Cys2/His2 zinc finger protein Zat7 have suppressed growth and are more tolerant to salinity stress. A deletion or a mutation of the EAR-motif of Zat7 abolishes salinity tolerance without affecting growth suppression. These results demonstrate that the EAR-motif of Zat7 is directly involved in enhancing the tolerance of transgenic plants to salinity stress. In contrast, the EAR-motif appears not to be involved in suppressing the growth of transgenic plants. Further analysis of Zat7 using RNAi lines suggests that Zat7 functions in Arabidopsis to suppress a repressor of defense responses. A yeast two-hybrid analysis identified putative interactors of Zat7 and the EAR-domain, including WRKY70 and HASTY, a protein involved in miRNA transport. Our findings demonstrate that the EAR-domain of Cys2/His2-type zinc finger proteins plays a key role in the defense response of Arabidopsis to abiotic stresses.