Paloma Koprovski Menguer

Roles of plant metal tolerance proteins (MTP) in metal storage and potential use in biofortification strategies

Zinc (Zn) is an essential micronutrient for plants, playing catalytic or structural roles in enzymes, transcription factors, ribosomes, and membranes. In humans, Zn deficiency is the second most common mineral nutritional disorder, affecting around 30% of the worlds population. People living in poverty usually have diets based on milled cereals, which contain low Zn concentrations. Biofortification of crops is an attractive cost-effective solution for low mineral dietary intake. In order to increase the amounts of bioavailable Zn in crop edible portions, it is necessary to understand how plants take up, distribute, and store Zn within their tissues, as well as to characterize potential candidate genes for biotechnological manipulation. The metal tolerance proteins (MTP) were described as metal efflux transporters from the cytoplasm, transporting mainly Zn2+ but also Mn2+, Fe2+, Cd2+, Co2+, and Ni2+. Substrate specificity appears to be conserved in phylogenetically related proteins. MTPs characterized so far in plants have a role in general Zn homeostasis and tolerance to Zn excess; in tolerance to excess Mn and also in the response to iron (Fe) deficiency. More recently, the first MTPs in crop species have been functionally characterized. In Zn hyperaccumulator plants, the MTP1 protein is related to hypertolerance to elevated Zn concentrations. Here, we review the current knowledge on this protein family, as well as biochemical functions and physiological roles of MTP transporters in Zn hyperaccumulators and non-accumulators. The potential applications of MTP transporters in biofortification efforts are discussed.

Functional analysis of the rice vacuolar zinc transporter OsMTP1

Heavy metal homeostasis is maintained in plant cells by specialized transporters which compartmentalize or efflux metal ions, maintaining cytosolic concentrations within a narrow range. OsMTP1 is a member of the cation diffusion facilitator (CDF)/metal tolerance protein (MTP) family of metal cation transporters in Oryza sativa, which is closely related to Arabidopsis thaliana MTP1. Functional complementation of the Arabidopsis T-DNA insertion mutant mtp1-1 demonstrates that OsMTP1 transports Zn in planta and localizes at the tonoplast. When heterologously expressed in the yeast mutant zrc1 cot1, OsMTP1 complemented its Zn hypersensitivity and was also localized to the vacuole. OsMTP1 alleviated, to some extent, the Co sensitivity of this mutant, rescued the Fe hypersensitivity of the ccc1 mutant at low Fe concentrations, and restored growth of the Cd-hypersensitive mutant ycf1 at low Cd concentrations. These results suggest that OsMTP1 transports Zn but also Co, Fe, and Cd, possibly with lower affinity. Site-directed mutagenesis studies revealed two substitutions in OsMTP1 that alter the transport function of this protein. OsMTP1 harbouring a substitution of Leu82 to a phenylalanine can still transport low levels of Zn, with an enhanced affinity for Fe and Co, and a gain of function for Mn. A substitution of His90 with an aspartic acid completely abolishes Zn transport but improves Fe transport in OsMTP1. These amino acid residues are important in determining substrate specificity and may be a starting point for refining transporter activity in possible biotechnological applications, such as biofortification and phytoremediation.

Identification of Fe-excess-induced genes in rice shoots reveals a WRKY transcription factor responsive to Fe, drought and senescence.

Fe participates in several important reactions in plant metabolism. However, Fe homeostasis in plants is not completely understood, and molecular studies on Fe-excess stress are scarce. Rice (Oryza sativa L. ssp. indica) is largely cultivated in submerged conditions, where the extremely reductive environment can lead to severe Fe overload. In this work, we used representational difference analysis (RDA) to isolate sequences up-regulated in rice shoots after exposure to Fe-excess. We isolated 24 sequences which have putative functions in distinct cellular processes, such as transcription regulation (OsWRKY80), stress response (OsGAP1, DEAD-BOX RNA helicase), proteolysis (oryzain-α,rhomboid protein), photosynthesis (chlorophyll a/b binding protein), sugar metabolism (β glucosidase) and electron transport (NADH ubiquinone oxireductase). We show that the putative WRKY transcription factor OsWRKY80 is up-regulated in rice leaves, stems and roots after Fe-excess treatment. This up-regulation is also observed after dark-induced senescence and drought stress, indicating that OsWRKY80 could be a general stress-responsive gene. To our knowledge, this is the first report of an Fe-excess-induced transcription factor in plants.

Got to hide your Zn away: Molecular control of Zn accumulation and biotechnological applications

Zinc (Zn) is an essential micronutrient for all organisms, with key catalytic and structural functions. Zn deficiency in plants, common in alkaline soils, results in growth arrest and sterility. On the other hand, Zn can become toxic at elevated concentrations. Several studies revealed molecules involved with metal acquisition in roots, distribution within the plant and translocation to seeds. Transmembrane Zn transport proteins and Zn chelators are involved in avoiding its toxic effects. Plant species with the capacity to hyperaccumulate and hypertolerate Zn have been characterized. Plants that accumulate and tolerate high amounts of Zn and produce abundant biomass may be useful for phytoremediation, allowing cleaning of metal-contaminated soils. The study of Zn hyperaccumulators may provide indications of genes and processes useful for biofortification, for developing crops with high amounts of nutrients in edible tissues. Future research needs to focus on functional characterization of Zn transporters in planta, elucidation of Zn uptake and sensing mechanisms, and on understanding the cross-talk between Zn homeostasis and other physiological processes. For this, new research should use multidisciplinary approaches, combining traditional and emerging techniques, such as genome-encoded metal sensors and multi-element imaging, quantification and speciation using synchrotron-based methods.

ZINC-INDUCED FACILITATOR-LIKE family in plants: lineage-specific expansion in monocotyledons and conserved genomic and expression features among rice ( Oryza sativa ) paralogs

BackgroundDuplications are very common in the evolution of plant genomes, explaining the high number of members in plant gene families. New genes born after duplication can undergo pseudogenization, neofunctionalization or subfunctionalization. Rice is a model for functional genomics research, an important crop for human nutrition and a target for biofortification. Increased zinc and iron content in the rice grain could be achieved by manipulation of metal transporters. Here, we describe the ZINC-INDUCED FACILITATOR-LIKE (ZIFL) gene family in plants, and characterize the genomic structure and expression of rice paralogs, which are highly affected by segmental duplication.ResultsSequences of sixty-eight ZIFL genes, from nine plant species, were comparatively analyzed. Although related to MSF_1 proteins, ZIFL protein sequences consistently grouped separately. Specific ZIFL sequence signatures were identified. Monocots harbor a larger number of ZIFL genes in their genomes than dicots, probably a result of a lineage-specific expansion. The rice ZIFL paralogs were named OsZIFL1 to OsZIFL13 and characterized. The genomic organization of the rice ZIFL genes seems to be highly influenced by segmental and tandem duplications and concerted evolution, as rice genome contains five highly similar ZIFL gene pairs. Most rice ZIFL promoters are enriched for the core sequence of the Fe-deficiency-related box IDE1. Gene expression analyses of different plant organs, growth stages and treatments, both from our qPCR data and from microarray databases, revealed that the duplicated ZIFL gene pairs are mostly co-expressed. Transcripts of OsZIFL4, OsZIFL5, OsZIFL7, and OsZIFL12 accumulate in response to Zn-excess and Fe-deficiency in roots, two stresses with partially overlapping responses.ConclusionsWe suggest that ZIFL genes have different evolutionary histories in monocot and dicot lineages. In rice, concerted evolution affected ZIFL duplicated genes, possibly maintaining similar expression patterns between pairs. The enrichment for IDE1 boxes in rice ZIFL gene promoters suggests a role in Zn-excess and Fe-deficiency up-regulation of ZIFL transcripts. Moreover, this is the first description of the ZIFL gene family in plants and the basis for functional studies on this family, which may play important roles in Zn and Fe homeostasis in plants.

kNACking on heaven’s door: how important are NAC transcription factors for leaf senescence and Fe/Zn remobilization to seeds?

Senescence is a coordinated process where a plant, or a part of it, engages in programmed cell death to salvage nutrients by remobilizing them to younger tissues or to developing seeds. As Fe and Zn deficiency are the two major nutritional disorders in humans, increased concentration of these nutrients through biofortification in cereal grains is a long-sought goal. Recent evidences point to a link between the onset of leaf senescence and increased Fe and Zn remobilization. In wheat, one member of the NAC (NAM, ATAF, and CUC) transcription factor (TF) family (NAM-B1) has a major role in the process, probably regulating key genes for the early onset of senescence, which results in higher Fe and Zn concentrations in grains. In rice, the most important staple food for nearly half of the world population, the NAM-B1 ortholog does not have the same function. However, other NAC proteins are related to senescence, and could be playing roles on the same remobilization pathway. Thus, these genes are potential tools for biofortification strategies in rice. Here we review the current knowledge on the relationship between senescence, Fe and Zn remobilization and the role of NAC TFs, with special attention to rice. We also propose a working model for OsNAC5, which would act on the regulation of nicotianamine (NA) synthesis and metal–NA remobilization.

Accumulation of brachycerine, an antioxidant glucosidic indole alkaloid, is induced by abscisic acid, heavy metal, and osmotic stress in leaves of Psychotria brachyceras.

Psychotria brachyceras Muell. Arg. produces the antioxidant monoterpene indole alkaloid (MIA) brachycerine, which, besides retaining a glucose residue, has its terpenoid moiety derived not from secologanin, but probably from epiloganin, representing a new subclass of MIAs. In this work we showed that osmotic stress agents, such as sodium chloride, sorbitol and polyethylene glycol (PEG), induced brachycerine accumulation in leaf disks of P. brachyceras. Other oxidative stress inducers, such as exposure to aluminum and silver, also increased brachycerine content. Abscisic acid (ABA) treatment was shown to increase brachycerine yield, suggesting its involvement in brachycerine induction during osmotic stress. Ascorbate peroxidase activity was induced in PEG-treated leaf disks, whereas superoxide dismutase (SOD) activity remained unaltered. Assays with specific inhibitors of the cytosolic mevalonate (MVA) and plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP) pathways showed that the terpenoid moiety of brachycerine derived predominantly from the MEP pathway. These results suggest a potential involvement of brachycerine in plant defense against osmotic/oxidative stress damage, possibly contributing to detoxification of hydroxyl radical and superoxide anion as a SOD-like molecule.

From soil to seed: micronutrient movement into and within the plant

The ability of roots to obtain micronutrients from the soil and to deliver these to the aerial tissues—including seeds—is essen- tial to ensure that the shoot has the resources it needs to function effectively. However, plants need to control several steps during the journey from soil to seed, including uptake, transport, remo- bilization and storage. A better comprehension of the relative contribution of these processes, together with their overall coor- dination, is necessary for a more complete understanding of plant metal homeostasis and for the development of successful biofor- tification strategies. This Research Topic aims at addressing some ofthemostrecent advances inmicronutrientmovementfromsoil to seed and to provide an overview of different approaches that can be used to generate micronutrient-efficient and biofortified plants. Here, we highlight some of the major points arising from these reports.

Stress Signaling Responses in Plants

Plants undergo continuous exposure to various biotic and abiotic stresses in their natural environment. To survive under such conditions, plants exhibit stress tolerance or stress avoidance through acclimation and adaptation mechanisms that ultimately reestablish cellular and organismal homeostasis or reduce episodic shock effects. These abilities involve intricate and complex mechanisms of perception, transduction, and transmission of stress stimuli, allowing optimal response to environmental conditions. The perception of stimuli and their expansion in cells involves signaling molecules such as intracellular calcium and reactive oxygen species, which intensify the action of particular signaling pathways. To date, the molecular mechanisms that are involved in each stress have been revealed comparatively independently, and so our understanding of convergence points between biotic and abiotic stress signaling pathways remains rudimentary. However, recent studies have revealed several molecules as promising candidates for common players that are involved in crosstalk between stress signaling pathways. This special issue aimed to join original research and review articles related to understanding of plant responses to abiotic and biotic stress conditions, identifying novel players involved in plant responses to stress conditions, biotechnological strategies to increase plant tolerance to abiotic and biotic stresses, and understanding molecular interactions and crosstalks among different stress conditions. M. Nourbakhsh-Rey and M. Libault in “Decipher the Molecular Response of Plant Single Cell Types to Environmental Stresses” explore omic studies to understand the response of single cell types to environmental stresses in order to clearly depict the contribution of each cell type composing the sample in response to stress. Cellular complexity of entire organs masks cell-specific responses to environmental stresses and logically leads to the dilution of the molecular changes occurring in each cell type composing the tissue/organ/plant in response to the stress. Specifically, the authors highlight that combining one or two omic analyses to look at single cell system biology can provide more precise molecular characterization and more dynamic models of the interactions between the plant and its environment. J. Ren et al. in “Drought Tolerance Is Correlated with the Activity of Antioxidant Enzymes in Cerasus humilis Seedlings” describe the correlation between the activities of antioxidant enzymes and drought tolerance. By exploring drought-resistant and drought-susceptible C. humilis accessions, they compare the abilities of the contrasting genotypes to induce antioxidant defense under drought conditions. Their manuscript presents original data indicating that plants exhibiting a more efficient reactive oxygen species scavenging system in response to drought conditions have enhanced membrane protection, an ability that could be directly linked to their higher adaptation to drought. X. Tang et al. in “Reference Gene Selection for qPCR Normalization of Kosteletzkya virginica under Salt Stress” employed RT-qPCR to select the most stable reference gene of the perennial halophytic plant K. virginica under salinity stresses. The stable reference gene selected in this study will be very helpful for revealing the gene expression profiles of K. virginica under salt stress, allowing a better understanding of the salt-resistant mechanisms in halophyte plants. Y. Oono et al. in “Genome-Wide Transcriptome Analysis of Cadmium Stress in Rice” used RNAseq strategy to elucidate the molecular basis of the rice response to cadmium (Cd) stress, a widespread heavy metal pollutant that is highly toxic to living cells, negatively affecting nutrient uptake and homeostasis in plants. In this work, rice plants were hydroponically treated with low Cd concentrations, revealing novel Cd-responsive transporters by analyzing gene expression under different Cd concentrations. This study could help to develop novel strategies for improving tolerance to Cd exposure in rice and other cereal crops. J. S. Rohila et al. in “Leaf Proteome Analysis Reveals Prospective Drought and Heat Stress Response Mechanisms in Soybean” investigate the effect of drought, heat, and cooccurring drought and heat stresses in the leaf proteome of two contrasting soybean genotypes. The authors identified genes involved in photosynthesis that were differentially expressed during drought and heat stress conditions, suggesting that photosynthesis-related proteins could be affecting RuBisCO regulation, electron transport, and Calvin cycle during abiotic stress, which ultimately alter the carbon fixation in leaves. The authors discuss the role of heat shock-related proteins and ROS detoxification capacity via carbonic anhydrase as heat and drought tolerance mechanisms, respectively. J. M. Garcia-Mina et al. in “Involvement of Hormone- and ROS-Signaling Pathways in the Beneficial Action of Humic Substances on Plants Growing under Normal and Stressing Conditions” review our current knowledge about the mechanisms by which soil humus affects soil fertility. In particular, they discuss the relationships between two main signaling pathway families that are affected by humic substances within the plant: hormone- and ROS-mediated signaling pathways. The authors aim to integrate these events in a more comprehensive model and point out future research directions to unveil the complete mechanisms of regulation. W. Fang et al. in “Cloning and Expression Analysis of One Gamma-Glutamylcysteine Synthetase Gene (Hbγ-ECS1) in Latex Production in Hevea brasiliensis” isolated a γ-ECS gene from the rubber tree and investigated its function linked to thiol content in latex. To understand the relation between γ-ECS and thiols and to correlate these findings to latex flow, the authors conducted RT-qPCR analysis and found that the expression levels of Hbγ-ECS1 were induced by tapping and ethrel stimulation, and the expression was related to thiols content in the latex. When looking at long-term flowing latex, the gene was related to the duration of latex flow. This work may have important biotechnological applications, since rubber tree is a major commercial source of latex, which is used for rubber production. A. Vian et al. in “Plant Responses to High Frequency Electromagnetic Fields” conducted an original review, looking at the possible effects of HF-EMFs, which are increasingly present in the environment (due to, e.g., cell phones, Wi-Fi, and different types of connected devices) on different essential plant metabolic activities, such as reactive oxygen species metabolism, Krebs cycle, pentose phosphate pathway, chlorophyll content, and terpene emission. They found that not only are most of these pathways indeed modified by HF-EMF exposure, but also radiation brings about alterations in gene expression and plant growth. The authors propose to consider nonionizing HF-EMF radiation as a noninjurious, albeit influential environmental factor that induces significant changes in plant metabolism.

Molecular Bases of Iron Accumulation Towards the Development of Iron-Enriched Crops

Abstract More than two billion people suffer from iron (Fe) deficiency, and the development of Fe-enriched crops could help to alleviate this problem. Event though major advances in Fe uptake from soil, xylem and phloem loading and grain sink strength have been made during the last years, several gaps in the long way between soil and grains still need to be elucidated, in order to identify effective molecular targets to generate biofortified crops. Here we focus on the major bottlenecks for Fe accumulation in crops, the Fe ligands that regulate Fe trafficking and localization in different cells and tissues, and the transgenic approaches (single-gene and multiple-genes) already used to significantly increase Fe accumulation in grains. We use knowledge accumulated in the model species Arabidopsis thaliana as well as in crops such as rice and maize. Also, we discuss future potential targets that may help to develop high seed Fe crops.