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 world's 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.

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.

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.

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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.

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.

Oryza sativa, the common cultivated rice, is one of the most important crops for human consumption, but production is increasingly threatened by abiotic stresses. Although many efforts have resulted in breeding rice cultivars that are relatively tolerant to their local environments, climate changes and population increase are expected to soon call for new, fast generation of stress tolerant rice germplasm, and current within-species rice diversity might not be enough to overcome such needs. The Oryza genus contains other 23 wild species, with only Oryza glaberrima being also domesticated. Rice domestication was performed with a narrow genetic diversity, and the other Oryza species are a virtually untapped genetic resource for rice stress tolerance improvement. Here we review the origin of domesticated Oryza sativa from wild progenitors, the ecological and genomic diversity of the Oryza genus, and the stress tolerance variation observed for wild Oryza species, including the genetic basis underlying the tolerance mechanisms found. The summary provided here is important to indicate how we should move forward to unlock the full potential of these germplasms for rice improvement.

SummaryZinc (Zn) is essential for all life forms, including humans. It is estimated that around two billion people are deficient in their Zn intake. Human dietary Zn intake relies heavily on plants, which in many developing countries consists mainly of cereals. The inner part of cereal grain, the endosperm, is the part that is eaten after milling but contains only a quarter of the total grain Zn. Here, we present results demonstrating that endosperm Zn content can be enhanced through expression of a transporter responsible for vacuolar Zn accumulation in cereals. The barley (Hordeum vulgare) vacuolar Zn transporter HvMTP1 was expressed under the control of the endosperm‐specific D‐hordein promoter. Transformed plants exhibited no significant change in growth but had higher total grain Zn concentration, as measured by ICP‐OES, compared to parental controls. Compared with Zn, transformants had smaller increases in concentrations of Cu and Mn but not Fe. Staining grain cross sections with the Zn‐specific stain DTZ revealed a significant enhancement of Zn accumulation in the endosperm of two of three transformed lines, a result confirmed by ICP‐OES in the endosperm of dissected grain. Synchrotron X‐ray fluorescence analysis of longitudinal grain sections demonstrated a redistribution of grain Zn from aleurone to endosperm. We argue that this proof‐of‐principle study provides the basis of a strategy for biofortification of cereal endosperm with Zn.