A path toward concurrent biofortification and cadmium mitigation in plant-based foods.

Millions of people are anemic due to inadequate consumption of foods rich in iron and zinc. Plant-based foods provide most of our dietary nutrients but may also contain the toxic heavy metal cadmium (Cd). A low level of Cd silently enters the body through the diet. Once ingested, Cd may remain for decades. Hence, prolonged intake of Cd-containing foods endangers human health. Research that leads towards micronutrient enrichment and mitigation of Cd in foods has therefore dual significance for human health. The breeding of Cd-tolerant cultivars may enable them to grow on Cd-polluted soils; however, they may not yield Cd-free foods. Conversely, sequestration of Cd in roots can prevent its accumulation in grains, but this mechanism also retains nutrients, hence counteracting biofortification efforts. A specific restriction of the Cd absorption capacity of crops would prevent Cd entry into the plant system while maintaining micronutrient accumulation and may thus be a solution to the dilemma. After recapitulating existing strategies employed for the development of Cd-tolerant and biofortified cultivars, this Viewpoint elaborates alternative approaches based on directed evolution and genome editing strategies for excluding Cd while enriching micronutrients in plant foods, which will concurrently help to eradicate malnutrition and prevent Cd intoxication.

Histone H3 lysine4 trimethylation-regulated GRF11 expression is essential for the iron-deficiency response in Arabidopsis thaliana.

Iron (Fe) homeostasis in plants is controlled by both transcription factors (TFs) and chromatin remodeling through histone modification. To date, few studies have reported the existence of histone modification in maintaining the Fe-deficiency response. However, the reports that do exist shed light on various histone modifications, but knowledge of the activation mark in Fe-deficiency response is lacking. By using a forward genetics approach, we identified a crucial allele for Fe-deficiency response, NON-RESPONSE TO Fe-DEFICIENCY2 (NRF2), previously described as EARLY FLOWERING8 (ELF8) associated with an activation mark on histone modification, histone H3 lysine4 trimethylation. In the nrf2-1 mutant, a point mutation at ELF8T404I , exhibits impaired expression of GENERAL REGULATORY FACTOR11 (GRF11) and downstream genes in the Fe-uptake pathway. In vivo chromatin immunoprecipitation revealed that in roots, NRF2/ELF8 is essential for the expression of GRF11 for Fe-deficiency response, whereas in shoots, NRF2/ELF8 regulates FLOWERING LOCUS C (FLC) expression for flowering time control. In summary, a key factor, NRF2/ELF8, involved in epigenetic regulation essential for both flowering time control and Fe-deficiency response is uncovered.

A HemK class glutamine-methyltransferase is involved in the termination of translation and essential for iron homeostasis in Arabidopsis.

Iron (Fe) transport and utilization are controlled by Fe-dependent transcriptional cascades. Many genes participate in these processes, transcriptionally controlled by Fe-status. Thorough knowledge of the translational check-points is lacking. We identified a non-response to Fe-deficiency1-1 (nrf1-1) mutant of Arabidopsis thaliana, which displayed a hypersensitive phenotype under Fe-deficient conditions. By mapping nrf1-1, we found that the AT3G13440 locus encoding a HemK methyltransferase is responsible for the phenotype. Analyses of ProUBQ10:NRF1CDS overexpression nrf1-1 lines and a T-DNA insertion mutant nrf1-2, confirmed that loss-of-function of NRF1 results in enhanced Fe-starvation-sensitivity. NRF1 is required for the proper expression of the majority of Fe-deficiency-inducible (FDI) genes. The nrf1 mutants accumulated more polysomes in the roots, due to stalled ribosomes on several transcripts. Ribosome-footprint (RF) mapping revealed that ribosomes are stalled at a stop codon that amplified the stalling of trailing ribosomes. We detected higher RF levels in many FDI transcripts in nrf1-2. Our study demonstrates the requirement of NRF1 for an accurate termination of protein synthesis essential not only for a precise iron homeostasis, but also cellular ion balance. NRF1 is also important for normal growth and development. A check-point that fine-tunes peptide release in plants is uncovered.

S-Nitrosoglutathione works downstream of nitric oxide to mediate iron-deficiency signaling in Arabidopsis.

Iron (Fe) is essential for plant growth and development. Knowledge of Fe signaling, from the beginning of perception to activation of the uptake process, is critical for crop improvement. Here, by using chemical screening, we identified a small molecule 3-amino-N-(3-methylphenyl)thieno[2,3-b]pyridine-2-carboxamide named R7 ('R' denoting repressor of IRON-REGULATED TRANSPORTER 1), that modulates Fe homeostasis of Arabidopsis. R7 treatment led to reduced Fe levels in plants, thus causing severe chlorosis under Fe deficiency. Expression analysis of central transcription factors, FER-LIKE IRON DEFICIENCY INDUCED TRANSCRIPTION FACTOR (FIT) and subgroup Ib basic helix-loop-helix (Ib bHLH) genes bHLH38/39/100/101, revealed that R7 targets the FIT-dependent transcriptional pathway. Exogenously supplying S-nitrosoglutathione (GSNO), but not other nitric oxide (NO) donors sodium nitroprusside (SNP) and S-nitroso-N-acetyl-dl-penicillamine (SANP), alleviated the inhibitory effects of R7 on Fe homeostasis. R7 did not inhibit cellular levels of NO or glutathione but decreased GSNO level in roots. We demonstrate that NO is involved in regulating not only the FIT transcriptional network but also the Ib bHLH networks. In addition, GSNO, from S-nitrosylation of glutathione, specifically mediates the Fe-starvation signal to FIT, which is distinct from the NO to Ib bHLH signal. Our work dissects the molecular connection between NO and the Fe-starvation response. We present a new signaling route whereby GSNO acts downstream of NO to trigger the Fe-deficiency response in Arabidopsis.

Small-Molecules Selectively Modulate Iron-Deficiency Signaling Networks in Arabidopsis.

Plant growth requires optimal levels of iron (Fe). Fe is used for energy production, numerous enzymatic processes, and is indispensable for cellular metabolism. Recent studies have established the mechanism involved in Fe uptake and transport. However, our knowledge of Fe sensing and signaling is limited. Dissecting Fe signaling may be useful for crop improvement by Fe fortification. Here, we report two small-molecules, R3 and R6 [where R denotes repressor of IRON-REGULATED TRANSPORTER 1 (IRT1)], identified through a chemical screening, whose use blocked activation of the Fe-deficiency response in Arabidopsis thaliana. Physiological analysis of plants treated with R3 and R6 showed that these small molecules drastically attenuated the plant response to Fe starvation. Small-molecule treatment caused severe chlorosis and strongly reduced chlorophyll levels in plants. Fe content in shoots was decreased considerably by small-molecule treatments especially in Fe deficiency. Small-molecule treatments attenuated the Fe-deficiency-induced expression of the Fe uptake gene IRT1. Analysis of FER-LIKE IRON-DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT) and subgroup Ib basic helix-loop-helix (bHLH) gene (bHLH38/39/100/101) expression showed that R3 affects the FIT-network, whereas R6 affects both the FIT and Ib bHLH networks. An assessment of the effects of the structural analogs of R3 and R6 on the induction of Fe-dependent chlorosis revealed the functional motif of the investigated chemicals. Our findings suggest that small-molecules selectively modulate the distinct signaling routes that operate in response to Fe-deficiency. R3 and R6 likely interrupt the activity of key upstream signaling regulators whose activities are required for the activation of the Fe-starvation transcriptional cascade in Arabidopsis roots.