Enhanced expression of OsNAC5 leads to up-regulation of OsNAC6 and changes rice (Oryza sativa L.) ionome.

NAC transcription factors are plant-specific proteins involved in many processes during the plant life cycle and responses to biotic and abiotic stresses. Previous studies have shown that stress-induced OsNAC5 from rice (Oryza sativa L.) is up-regulated by senescence and might be involved in control of iron (Fe) and zinc (Zn) concentrations in rice seeds. Aiming a better understanding of the role of OsNAC5 in rice plants, we investigated a mutant line carrying a T-DNA insertion in the promoter of OsNAC5, which resulted in enhanced expression of the transcription factor. Plants with OsNAC5 enhanced expression were shorter at the seedling stage and had reduced yield at maturity. In addition, we evaluated the expression level of OsNAC6, which is co-expressed with OsNAC5, and found that enhanced expression of OsNAC5 leads to increased expression of OsNAC6, suggesting that OsNAC5 might regulate OsNAC6 expression. Ionomic analysis of leaves and seeds from the OsNAC5 enhanced expression line revealed lower Fe and Zn concentrations in leaves and higher Fe concentrations in seeds than in WT plants, further suggesting that OsNAC5 may be involved in regulating the ionome in rice plants. Our work shows that fine-tuning of transcription factors is key when aiming at crop improvement.

A tale of two metals: Biofortification of rice grains with iron and zinc.

Iron (Fe) and zinc (Zn) are essential micronutrients needed by virtually all living organisms, including plants and humans, for proper growth and development. Due to its capacity to easily exchange electrons, Fe is important for electron transport in mitochondria and chloroplasts. Fe is also necessary for chlorophyll synthesis. Zn is a cofactor for several proteins, including Zn-finger transcription factors and redox metabolism enzymes such as copper/Zn superoxide dismutases. In humans, Fe participates in oxygen transport, electron transport, and cell division whereas Zn is involved in nucleic acid metabolism, apoptosis, immunity, and reproduction. Rice (Oryza sativa L.) is one of the major staple food crops, feeding over half of the world's population. However, Fe and Zn concentrations are low in rice grains, especially in the endosperm, which is consumed as white rice. Populations relying heavily on rice and other cereals are prone to Fe and Zn deficiency. One of the most cost-effective solutions to this problem is biofortification, which increases the nutritional value of crops, mainly in their edible organs, without yield reductions. In recent years, several approaches were applied to enhance the accumulation of Fe and Zn in rice seeds, especially in the endosperm. Here, we summarize these attempts involving transgenics and mutant lines, which resulted in Fe and/or Zn biofortification in rice grains. We review rice plant manipulations using ferritin genes, metal transporters, changes in the nicotianamine/phytosiderophore pathway (including biosynthetic genes and transporters), regulators of Fe deficiency responses, and other mutants/overexpressing lines used in gene characterization that resulted in Fe/Zn concentration changes in seeds. This review also discusses research gaps and proposes possible future directions that could be important to increase the concentration and bioavailability of Fe and Zn in rice seeds without the accumulation of deleterious elements. We also emphasize the need for a better understanding of metal homeostasis in rice, the importance of evaluating yield components of plants containing transgenes/mutations under field conditions, and the potential of identifying genes that can be manipulated by gene editing and other nontransgenic approaches.

Identification of Promising Genotypes Through Systematic Evaluation for Arsenic Tolerance and Exclusion in Rice (Oryza sativa L.).

Rice remains a major staple food source for the rapidly growing world population. However, regular occurrences of carcinogenic arsenic (As) minerals in waterlogged paddy topsoil pose a great threat to rice production and consumers across the globe. Although As contamination in rice has been well recognized over the past two decades, no suitable rice germplasm had been identified to exploit in adaptive breeding programs. Therefore, this current study identified suitable rice germplasm for As tolerance and exclusion based on a variety of traits and investigated the interlinkages of favorable traits during different growth stages. Fifty-three different genotypes were systematically evaluated for As tolerance and accumulation. A germination screening assay was carried out to identify the ability of individual germplasm to germinate under varying As stress. Seedling-stage screening was conducted in hydroponics under varying As stress to identify tolerant and excluder genotypes, and a field experiment was carried out to identify genotypes accumulating less As in grain. Irrespective of the rice genotypes, plant health declined significantly with increasing As in the treatment. However, genotype-dependent variation in germination, tolerance, and As accumulation was observed among the genotypes. Some genotypes (WTR1-BRRI dhan69, NPT-IR68552-55-3-2, OM997, and GSR IR1-5-Y4-S1-Y1) showed high tolerance by excluding As in the shoot system. Arsenic content in grain ranged from 0.12 mg kg-1 in Huang-Hua-Zhan (indica) from China to 0.48 mg kg-1 in IRAT 109 (japonica) from Brazil. This current study provides novel insights into the performance of rice genotypes under varying As stress during different growth stages for further use in ongoing breeding programs for the development of As-excluding rice varieties for As-polluted environments.

Chromosomal introgressions from Oryza meridionalis into domesticated rice Oryza sativa result in iron tolerance.

Iron (Fe) toxicity is one of the most common mineral disorders affecting rice (Oryza sativa) production in flooded lowland fields. Oryza meridionalis is indigenous to northern Australia and grows in regions with Fe-rich soils, making it a candidate for use in adaptive breeding. With the aim of understanding tolerance mechanisms in rice, we screened a population of interspecific introgression lines from a cross between O. sativa and O. meridionalis for the identification of quantitative trait loci (QTLs) contributing to Fe-toxicity tolerance. Six putative QTLs were identified. A line carrying one introgression from O. meridionalis on chromosome 9 associated with one QTL was highly tolerant despite very high shoot Fe concentrations. Physiological, biochemical, ionomic, and transcriptomic analyses showed that the tolerance of the introgression lines could partly be explained by higher relative Fe retention in the leaf sheath and culm. We constructed the interspecific hybrid genome in silico for transcriptomic analysis and identified differentially regulated introgressed genes from O. meridionalis that could be involved in shoot-based Fe tolerance, such as metallothioneins, glutathione S-transferases, and transporters from the ABC and MFS families. This work demonstrates that introgressions of O. meridionalis into the O. sativa genome can confer increased tolerance to excess Fe.

The Mitochondrial Iron-Regulated (MIR) gene is Oryza genus specific and evolved before speciation within the Oryza sativa complex.

MAIN CONCLUSION: The MIR gene is not an Oryza sativa orphan gene, but an Oryza genus-specific gene that evolved before AA lineage speciation by a complex origination process. Rice (Oryza sativa L.) is a model species and an economically relevant crop. The Oryza genus comprises 25 species, with genomic data available for several Oryza species, making it a model for genetics and evolution. The Mitochondrial Iron-Regulated (MIR) gene was previously implicated in the O. sativa Fe deficiency response, and was considered an orphan gene present only in rice. Here we show that MIR is also found in other Oryza species that belong to the Oryza sativa complex, which have AA genome type and constitute the primary gene pool for O. sativa breeding. Our data suggest that MIR originated in a stepwise process, in which sequences derived from an exon fragment of the raffinose synthase gene were pseudogenized into non-coding, which in turn originated the MIR gene de novo. All species with a putative functional MIR gene conserve their regulation by Fe deficiency, with the exception of Oryza barthii. In O. barthii, the MIR coding sequence was translocated to a different chromosomal position and separated from its regulatory region, leading to a lack of Fe deficiency responsiveness. Moreover, the MIR co-expression subnetwork cluster in O. sativa is responsive to Fe deficiency, evidencing the importance of the newly originated gene in Fe uptake. This work establishes that MIR is not an orphan gene as previously proposed, but a de novo originated gene within the genus Oryza. We also showed that MIR is undergoing genomic changes in one species (O. barthii), with an impact on Fe deficiency response.

The Combined Strategy for iron uptake is not exclusive to domesticated rice (Oryza sativa).

Iron (Fe) is an essential micronutrient that is frequently inaccessible to plants. Rice (Oryza sativa L.) plants employ the Combined Strategy for Fe uptake, which is composed by all features of Strategy II, common to all Poaceae species, and some features of Strategy I, common to non-Poaceae species. To understand the evolution of Fe uptake mechanisms, we analyzed the root transcriptomic response to Fe deficiency in O. sativa and its wild progenitor O. rufipogon. We identified 622 and 2,017 differentially expressed genes in O. sativa and O. rufipogon, respectively. Among the genes up-regulated in both species, we found Fe transporters associated with Strategy I, such as IRT1, IRT2 and NRAMP1; and genes associated with Strategy II, such as YSL15 and IRO2. In order to evaluate the conservation of these Strategies among other Poaceae, we identified the orthologs of these genes in nine species from the Oryza genus, maize and sorghum, and evaluated their expression profile in response to low Fe condition. Our results indicate that the Combined Strategy is not specific to O. sativa as previously proposed, but also present in species of the Oryza genus closely related to domesticated rice, and originated around the same time the AA genome lineage within Oryza diversified. Therefore, adaptation to Fe2+ acquisition via IRT1 in flooded soils precedes O. sativa domestication.

Potassium Ion Channel Gene OsAKT1 Affects Iron Translocation in Rice Plants Exposed to Iron Toxicity.

Iron toxicity is one of the most widely spread mineral disorders in anaerobic soils, but the tolerance mechanisms in plants are poorly understood. Here we characterize the involvement of a rice potassium ion channel gene, OsAKT1, in Fe toxic conditions. Two knock-down lines of OsAKT1 together with azygos lines were investigated. Mutant lines did not differ from azygos lines regarding plant growth, gas exchange rate or chlorophyll fluorescence in control conditions. However, loss-of-function of OsAKT1 increased the sensitivity to excess Fe regarding leaf bronzing symptoms, reactive oxygen species generation, leaf spectral reflectance indices, and chlorophyll fluorescence. Fe toxicity leads to largely reduced uptake of other nutrients into shoots, which illustrates the complexity of Fe stress related to multiple mineral disorders. Less potassium uptake in the mutants compared to azygos lines co-occurred with higher amounts of Fe accumulated in the shoot tissues but not in the roots. These results were consistent with a higher level of Fe loaded into the xylem sap of mutants compared to azygos lines in the early phase of Fe toxicity. In conclusion, OsAKT1 is crucial for the tolerance of rice against Fe toxicity as K homeostasis affects Fe translocation from root to shoot.