Naoko K. Nishizawa

Iron is an essential element for organisms, but its solubility in soil is often extremely low. Previously, plants were considered to take up iron only after its reduction to ferrous ions. Takagi reported that oat and rice secrete chelating substances that solubilize ferric iron in the rhizosphere for efficient iron uptake. In 1978, Takemoto et al. reported the chemical structure of an iron-chelating compound secreted from barley roots, designated as mugineic acid. Mugineic acid and its derivatives, collectively known as mugineic acid family phytosiderophores (MAs), chelate ferric iron using octahedral hexacoordination. The specific iron uptake system by MAs in graminaceous plants was later classified by Römheld and Marschner as Strategy II, in contrast to Strategy I for reduction-based iron uptake by non-graminaceous plants. Further studies on MAs by Japanese researchers led to the identification of their biosynthetic pathways, corresponding enzymes and encoding genes, their regulation mechanisms, and the production of iron deficiency-tolerant and iron-rich crops.

Iron (Fe) is an essential micronutrient for plants. Plants encounter Fe deficiency when grown in calcareous soil with low Fe availability, leading to reduced crop yield and agricultural problem. Rice acquires Fe from the soil via Strategy I–related system (ferrous ion uptake by OsIRT1) and Strategy II system (ferric ion uptake by chelation). However, rice plants have a weak ability in Fe(III) reduction and phytosiderophore secretion. We previously produced an Fe deficiency–tolerant rice harboring OsIRT1 promoter-refre1/372 (for higher Fe(III) reductase ability) and a 35S promoter-OsIRO2 (for higher phytosiderophore secretion). In this study, we produced a new Fe deficiency–tolerant rice by the additional introduction of a barley IDS3 genome fragment with refre1/372 and OsIRO2 (named as IRI lines) for further enhancement in Strategy II phytosiderophore productivity and better growth performance in various environments. Our results show that an enhanced tolerance was observed in OsIRO2 introduced line at the early growth stage, refre1/372 introduced line in the late stage, and RI line in all stages among five types of cultivation method. Moreover, we demonstrated that new IRI rice lines exhibited enhanced tolerance to Fe deficiency compared to nontransgenic (NT) rice and rice lines harboring the overexpressing OsIRO2 or the IDS3 fragment under submerged calcareous soil. The yields of IRI lines were ninefold higher than the NT line. Furthermore, under Fe-limited nonsubmerged calcareous soil condition (a new cultivation condition), IRI lines also conferred enhanced tolerance than NT, lines introducing only the OsIRT1 promoter-refre1/372 or overexpressing OsIRO2, and lines harboring both. Our results demonstrate that further enhancement of the Strategy II Fe uptake system by the mugineic acid synthase gene in addition to Fe uptake by enhanced ferric Fe reduction and phytosiderophore production in rice contributes Fe deficiency tolerance and broaden its utility in calcareous soil cultivation under paddy or nonpaddy field conditions.

Iron (Fe) toxicity in plants causes tissue damage and cellular homeostasis disorders, thereby affecting plant growth and development. Nicotianamine (NA) is a ubiquitous chelator of metal cations and is responsible for metal homeostasis. Rice has three NA synthase (NAS) genes, of which the expression of OsNAS1 and OsNAS2 but not of OsNAS3 is strongly induced in response to Fe deficiency. Recently, we found that OsNAS3 expression is strongly induced with excess Fe in most rice tissues, particularly old leaves, suggesting that it may play a vital role under excess Fe conditions. However, the mechanism by which OsNAS3 responds to excess Fe in rice remains poorly understood. In this study, we clarified the physiological response of OsNAS3 expression to excess Fe and the role of NA synthesis in this condition. Promoter GUS analyses revealed that OsNAS3 was widely expressed in roots, especially in vascular bundle, epidermis, exodermis, stem, and old leaf tissues under Fe excess compared to control plants. Nicotianamine and deoxymugineic acid (DMA; a type of phytosiderophore synthesized by Strategy II species) were present in roots and shoots under Fe excess likewise under control conditions. In addition, OsNAS3 knockout plants were sensitive to excess Fe, exhibiting inferior growth, reduced dry weight, severer leaf bronzing, and greater Fe accumulation in their leaves than non-transformants with excess Fe. We also observed that NA-overproducing rice was tolerant of excess Fe. These results show that NA synthesized by OsNAS3 under Fe excess condition is to mitigate excess Fe whereas NA synthesized by OsNAS1 and OsNAS2 under normal Fe condition is to enhance Fe translocation, suggesting the different roles and functions of the NA existence between these two conditions. Overall, these findings suggest that rice synthesizes NA with OsNAS3 under Fe excess in roots and shoots, and that NA and DMA within the plant body are important for mitigating excess Fe stress and alleviating other metal deficiencies in rice. This report will be important for the development of tolerant rice adapted to Fe-contaminated soils.

Copper (Cu) is involved in fundamental biological processes for plant growth and development. However, Cu excess is harmful to plants. Thus, Cu in plant tissues must be tightly regulated. In this study, we found that the peanut Yellow Stripe-Like family gene AhYSL3.1 is involved in Cu transport. Among five AhYSL genes, AhYSL3.1 and AhYSL3.2 were upregulated by Cu deficiency in peanut roots and expressed mainly in young leaves. A yeast complementation assay suggested that the plasma membrane-localized AhYSL3.1 was a Cu-nicotianamine complex transporter. High expression of AhYSL3.1 in tobacco and rice plants with excess Cu resulted in a low concentration of Cu in young leaves. These transgenic plants were resistant to excess Cu. The above results suggest that AhYSL3.1 is responsible for the internal transport of Cu in peanut.

Iron (Fe) deficiency is a serious agricultural problem, especially in calcareous soils, which are distributed worldwide. Poplar trees are an important biomass plant, and overcoming Fe deficiency in poplars will increase biomass productivity worldwide. The poplar Fe-deficiency response and the genes involved in poplar Fe homeostasis remain largely unknown. To identify these genes and processes, we cultivated poplar plants under Fe-deficient conditions, both in calcareous soil and hydroponically, and analyzed their growth rates, leaf Soil and Plant Analyzer Development (SPAD) values, and metal concentrations. The data clearly showed that poplars have notable growth defects in both calcareous soil and a Fe-deficient hydroponic culture. They exhibited serious chlorosis of young leaves after 3 weeks of Fe-deficient hydroponic culture. The Fe concentrations in old leaves with high SPAD values were markedly lower in Fe-deficient poplars, suggesting that poplars may have good translocation capability from old to new leaves. The Zn concentration in new leaves increased in Fe-deficient poplars. The pH of the hydroponic solution decreased in the Fe-deficient culture compared to the Fe-sufficient culture. This finding shows that poplars may be able to adjust the pH of a culture solution to better take up Fe. We also analyzed the expression of Fe homeostasis-related genes in the roots and leaves of Fe-sufficient and Fe-deficient poplars. Our results demonstrate that PtIRT1, PtNAS2, PtFRO2, PtFRO5, and PtFIT were induced in Fe-deficient roots. PtYSL2 and PtNAS4 were induced in Fe-deficient leaves. PtYSL3 was induced in both Fe-deficient leaves and roots. These genes may be involved in the Fe uptake and/or translocation mechanisms in poplars under Fe-deficient conditions. Our results will increase a better understanding of the Fe-deficiency response of poplars and hence improve the breeding of Fe-deficiency-tolerant poplars for improved biomass production, the greening of high pH soils, and combatting global warming.

Iron is essential for virtually all organisms but is toxic when present in excess. To acquire the proper amount of iron, plants induce expression of various genes involved in iron uptake and translocation in response to low‐iron availability. Two iron‐binding ubiquitin ligases, OsHRZ1 and OsHRZ2, negatively regulate such iron deficiency responses in rice (Oryza sativa). Transgenic rice plants with repressed expression of OsHRZ1 and OsHRZ2 (HRZ‐knockdown lines) are tolerant to low iron availability and accumulate iron in shoots and seeds under both iron‐sufficient and iron‐deficient conditions without a growth penalty. Although the expression of OsHRZ1 and OsHRZ2 is transcriptionally upregulated under iron‐deficient conditions, the physiological relevance of this induction is not known. In the present study, we analyzed the response of HRZ‐knockdown lines to excess iron. In the presence of severe excess iron, the HRZ‐knockdown lines grew worse than non‐transformants (NTs). The HRZ‐knockdown lines showed stunted shoot and root growth and more severe leaf bronzing compared to NTs. Moreover, these lines accumulated more iron in shoots and exhibited severely elevated expression of various genes involved in iron uptake and translocation as well as jasmonate signaling compared to NTs. These results indicate that HRZ ubiquitin ligases are crucial for repressing iron deficiency responses and protecting cells from iron toxicity in the presence of excess iron. These results support the possibility that HRZs are intracellular Fe sensors and provide clues for developing plants tolerant of either iron deficiency or excess with higher iron contents in edible parts.

Iron (Fe) toxicity is a major nutritional disorder of plants and affects rice yield and production in rainfed and irrigated lowland rice grown in acid soils. Rice plants are reported to have exclusion and inclusion adaptation strategies for preventing damage from excess Fe. However, the molecular mechanisms behind the Fe toxicity response and the identities of the genes involved remain largely unknown. To reveal these mechanisms, we exposed rice plants to different levels of ferrous (Fe²⁺) excess treatment for 14 days and analyzed their growth, bronzing score, and mineral concentrations. Then, gene expression patterns in various tissues (roots, discrimination center [DC], stems, old leaves [OLs], and newest leaves [NLs]) in response to different levels of Fe excess (×1, ×10, ×20, ×50, and ×70 Fe) were examined using microarray analysis. Our results showed that the higher levels of Fe excess led to more Fe being preferentially translocated to OLs, thus avoiding Fe excess damage in the NL. We proposed three zones of Fe excess levels: the non-affected, affected, and dead zones. As an exclusion strategy, Fe uptake- and transport-related genes were suppressed in roots since in the non-affected zone. Roots are important for preventing Fe uptake to the plant body under Fe excess stress. As inclusion strategies, first, some genes highly induced in various tissues under Fe excess, such as OsNAS3, OsVIT2, and rice ferritin genes (OsFers), may be important for detoxification or isolation of excess Fe within the plant body. OsZIPs may contribute to the maintenance of zinc homeostasis. Second, the plant induces the expression of oxygen and electron transfer genes, cytochrome P450 family proteins, or some NAC-type transcription factors to avoid reactive oxygen species and abiotic stress caused by Fe excess in the affected zone. The plant may use similar Fe homeostasis mechanisms in the non-affected and affected zones in the NL and roots but employ different mechanisms in the OL, DC, and stem tissues. Our results will contribute to current screening and breeding efforts, which aim to develop Fe excess tolerance in diverse rice cultivars, thus increasing rice production in lowland fields.

Rice OsYSL9 is a novel transporter for Fe(II)-nicotianamine and Fe(III)-deoxymugineic acid that is responsible for internal iron transport, especially from endosperm to embryo in developing seeds.