Rice is the primary source of food for billions of people in developing countries, yet the commonly consumed polished grain contains insufficient levels of the key micronutrients iron (Fe), zinc (Zn) and Vitamin A to meet daily dietary requirements. Experts estimate that a rice-based diet should contain 14.5 µg g⁻¹ Fe in endosperm, the main constituent of polished grain, but breeding programs have failed to achieve even half of that value. Transgenic efforts to increase the Fe concentration of rice endosperm include expression of ferritin genes, nicotianamine synthase genes (NAS) or ferritin in conjunction with NAS genes, with results ranging from two-fold increases via single-gene approaches to six-fold increases via multi-gene approaches, yet no approach has reported 14.5 µg g⁻¹ Fe in endosperm. Three populations of rice were generated to constitutively overexpress OsNAS1, OsNAS2 or OsNAS3, respectively. Nicotianamine, Fe and Zn concentrations were significantly increased in unpolished grain of all three of the overexpression populations, relative to controls, with the highest concentrations in the OsNAS2 and OsNAS3 overexpression populations. Selected lines from each population had at least 10 µg g⁻¹ Fe in polished grain and two OsNAS2 overexpression lines had 14 and 19 µg g⁻¹ Fe in polished grain, representing up to four-fold increases in Fe concentration. Two-fold increases of Zn concentration were also observed in the OsNAS2 population. Synchrotron X-ray fluorescence spectroscopy demonstrated that OsNAS2 overexpression leads to significant enrichment of Fe and Zn in phosphorus-free regions of rice endosperm. The OsNAS genes, particularly OsNAS2, show enormous potential for Fe and Zn biofortification of rice endosperm. The results demonstrate that rice cultivars overexpressing single rice OsNAS genes could provide a sustainable and genetically simple solution to Fe and Zn deficiency disorders affecting billions of people throughout the world.

More than two billion people are micronutrient deficient. Polished grains of popular rice varieties have concentration of approximately 2 μg g⁻¹ iron (Fe) and 16 μg g⁻¹zinc (Zn). The HarvestPlus breeding programs for biofortified rice target 13 μg g⁻¹Fe and 28 μg g⁻¹Zn to reach approximately 30% of the estimated average requirement (EAR). Reports on engineering Fe content in rice have shown an increase up to 18 μg g⁻¹ in glasshouse settings; in contrast, under field conditions, 4 μg g⁻¹ was the highest reported concentration. Here, we report on selected transgenic events, field evaluated in two countries, showing 15 μg g⁻¹Fe and 45.7 μg g⁻¹Zn in polished grain. Rigorous selection was applied to 1,689 IR64 transgenic events for insert cleanliness and, trait and agronomic performances. Event NASFer-274 containing rice nicotianamine synthase (OsNAS2) and soybean ferritin (SferH-1) genes showed a single locus insertion without a yield penalty or altered grain quality. Endosperm Fe and Zn enrichment was visualized by X-ray fluorescence imaging. The Caco-2 cell assay indicated that Fe is bioavailable. No harmful heavy metals were detected in the grain. The trait remained stable in different genotype backgrounds.

Previously, cell type-specific expression of AtHKT1;1, a sodium transporter, improved sodium (Na⁺) exclusion and salinity tolerance in Arabidopsis. In the current work, AtHKT1;1, was expressed specifically in the root cortical and epidermal cells of an Arabidopsis GAL4-GFP enhancer trap line. These transgenic plants were found to have significantly improved Na⁺ exclusion under conditions of salinity stress. The feasibility of a similar biotechnological approach in crop plants was explored using a GAL4-GFP enhancer trap rice line to drive expression of AtHKT1;1 specifically in the root cortex. Compared with the background GAL4-GFP line, the rice plants expressing AtHKT1;1 had a higher fresh weight under salinity stress, which was related to a lower concentration of Na⁺ in the shoots. The root-to-shoot transport of ²²Na⁺ was also decreased and was correlated with an upregulation of OsHKT1;5, the native transporter responsible for Na⁺ retrieval from the transpiration stream. Interestingly, in the transgenic Arabidopsis plants overexpressing AtHKT1;1 in the cortex and epidermis, the native AtHKT1;1 gene responsible for Na⁺ retrieval from the transpiration stream, was also upregulated. Extra Na⁺ retrieved from the xylem was stored in the outer root cells and was correlated with a significant increase in expression of the vacuolar pyrophosphatases (in Arabidopsis and rice) the activity of which would be necessary to move the additional stored Na⁺ into the vacuoles of these cells. This work presents an important step in the development of abiotic stress tolerance in crop plants via targeted changes in mineral transport.

Elevated salinity imposes osmotic and ion toxicity stresses on living cells and requires a multitude of responses in order to enable plant survival. Building on earlier work profiling transcript levels in rice (Oryza sativa) shoots of FL478, a salt-tolerant indica recombinant inbred line, and IR29, a salt-sensitive cultivar, transcript levels were compared in roots of these two accessions as well as in the roots of two additional salt-tolerant indica genotypes, the landrace Pokkali and the recombinant inbred line IR63731. The aim of this study was to compare transcripts in the sensitive and the tolerant lines in order to identify genes likely to be involved in plant salinity tolerance, rather than in responses to salinity per se. Transcript profiles of several gene families with known links to salinity tolerance are described (e.g. HKTs, NHXs). The putative function of a set of genes identified through their salt responsiveness, transcript levels, and/or chromosomal location (i.e. underneath QTLs for salinity tolerance) is also discussed. Finally, the parental origin of the Saltol region in FL478 is further investigated. Overall, the dataset presented appears to be robust and it seems likely that this system could provide a reliable strategy for the discovery of novel genes involved in salinity tolerance.

Under saline conditions, higher plants restrict the accumulation of chloride ions (Cl^–) in the shoot by regulating their transfer from the root symplast into the xylem-associated apoplast. To identify molecular mechanisms underpinning this phenomenon, we undertook a transcriptional screen of salt stressed Arabidopsis (Arabidopsis thaliana) roots. Microarrays, quantitative RT-PCR, and promoter-GUS fusions identified a candidate gene involved in Cl^– xylem loading from the Nitrate transporter 1/Peptide Transporter family (NPF2.4). This gene was highly expressed in the root stele compared to the cortex, and its expression decreased after exposure to NaCl or abscisic acid. NPF2.4 fused to fluorescent proteins, expressed either transiently or stably, was targeted to the plasma membrane. Electrophysiological analysis of NPF2.4 in Xenopus laevis oocytes suggested that NPF2.4 catalyzed passive Cl^– efflux out of cells and was much less permeable to NO₃⁻. Shoot Cl^– accumulation was decreased following NPF2.4 artificial microRNA knockdown, whereas it was increased by overexpression of NPF2.4. Taken together, these results suggest that NPF2.4 is involved in long-distance transport of Cl^– in plants, playing a role in the loading and the regulation of Cl^– loading into the xylem of Arabidopsis roots during salinity stress.

Abiotic stresses, such as drought, salinity, and extreme temperatures, are major limiting factors in global crop productivity and are predicted to be exacerbated by climate change. The overproduction of reactive oxygen species (ROS) is a common consequence of many abiotic stresses. Ascorbate, also known as vitamin C, is the most abundant water-soluble antioxidant in plant cells and can combat oxidative stress directly as a ROS scavenger, or through the ascorbate–glutathione cycle—a major antioxidant system in plant cells. Engineering crops with enhanced ascorbate concentrations therefore has the potential to promote broad abiotic stress tolerance. Three distinct strategies have been utilized to increase ascorbate concentrations in plants: (i) increased biosynthesis, (ii) enhanced recycling, or (iii) modulating regulatory factors. Here, we review the genetic pathways underlying ascorbate biosynthesis, recycling, and regulation in plants, including a summary of all metabolic engineering strategies utilized to date to increase ascorbate concentrations in model and crop species. We then highlight transgene-free strategies utilizing genome editing tools to increase ascorbate concentrations in crops, such as editing the highly conserved upstream open reading frame that controls translation of the GDP-L-galactose phosphorylase gene.