SUMMARYZinc is an essential micronutrient for plants, but it is toxic in excess concentrations. In Arabidopsis, additional iron (Fe) can increase Zn tolerance. We isolated a mutant, zinc tolerance induced by iron 1, designated zir1, with a defect in Fe-mediated Zn tolerance. Using map-based cloning and genetic complementation, we identified that zir1 has a mutation of glutamate to lysine at position 385 on c-glutamylcysteine synthetase (GSH1), the enzyme involved in glutathione biosynthesis. The zir1 mutant contains only 15% of the wild-type glutathione level. Blocking glutathione biosynthesis in wild-type plants by a specific inhibitor of GSH1, buthionine sulfoximine, resulted in loss of Fe-mediated Zn tolerance, which provides further evidence that glutathione plays an essential role in Fe-mediated Zn tolerance. Two glutathione-deficient mutant alleles of GSH1, pad2-1 and cad2-1, which contain 22% and 39%, respectively, of the wild-type glutathione level, revealed that a minimal glutathione level between 22 and 39% of the wild-type level is required for Fe-mediated Zn tolerance. Under excess Zn and Fe, the recovery of shoot Fe contents in pad2-1 and cad2-1 was lower than that of the wild type. However, the phytochelatin-deficient mutant cad1-3 showed normal Fe-mediated Zn tolerance. These results indicate a specific role of glutathione in Fe-mediated Zn tolerance. The induced accumulation of glutathione in response to excess Zn and Fe suggests that glutathione plays a specific role in Fe-mediated Zn tolerance in Arabidopsis. We conclude that glutathione is required for the cross-homeostasis between Zn and Fe in Arabidopsis.

Summary Iron (Fe) deficiency is a common agricultural problem that affects both the productivity and nutritional quality of plants. Thus, identifying the key factors involved in the tolerance of Fe deficiency is important. In the present study, the zir1 mutant, which is glutathione deficient, was found to be more sensitive to Fe deficiency than the wild type, and grew poorly in alkaline soil. Other glutathione‐deficient mutants also showed various degrees of sensitivity to Fe‐limited conditions. Interestingly, we found that the glutathione level was increased under Fe deficiency in the wild type. By contrast, blocking glutathione biosynthesis led to increased physiological sensitivity to Fe deficiency. On the other hand, overexpressing glutathione enhanced the tolerance to Fe deficiency. Under Fe‐limited conditions, glutathione‐deficient mutants, zir1, pad2 and cad2 accumulated lower levels of Fe than the wild type. The key genes involved in Fe uptake, including IRT1, FRO2 and FIT, are expressed at low levels in zir1; however, a split‐root experiment suggested that the systemic signals that govern the expression of Fe uptake‐related genes are still active in zir1. Furthermore, we found that zir1 had a lower accumulation of nitric oxide (NO) and NO reservoir S‐nitrosoglutathione (GSNO). Although NO is a signaling molecule involved in the induction of Fe uptake‐related genes during Fe deficiency, the NO‐mediated induction of Fe‐uptake genes is dependent on glutathione supply in the zir1 mutant. These results provide direct evidence that glutathione plays an essential role in Fe‐deficiency tolerance and NO‐mediated Fe‐deficiency signaling in Arabidopsis.

The homeostasis of iron (Fe) in plants is strictly regulated to maintain an optimal level for plant growth and development but not cause oxidative stress. About 30% of arable land is considered Fe deficient because of calcareous soil that renders Fe unavailable to plants. Under Fe-deficient conditions, Arabidopsis (Arabidopsis thaliana) shows retarded growth, disordered chloroplast development, and delayed flowering time. In this study, we explored the possible connection between Fe availability and the circadian clock in growth and development. Circadian period length in Arabidopsis was longer under Fe-deficient conditions, but the lengthened period was not regulated by the canonical Fe-deficiency signaling pathway involving nitric oxide. However, plants with impaired chloroplast function showed long circadian periods. Fe deficiency and impaired chloroplast function combined did not show additive effects on the circadian period, which suggests that plastid-to-nucleus retrograde signaling is involved in the lengthening of circadian period under Fe deficiency. Expression pattern analyses of the central oscillator genes in mutants defective in CIRCADIAN CLOCK ASSOCIATED1/LATE ELONGATED HYPOCOTYL or GIGANTEA demonstrated their requirement for Fe deficiency-induced long circadian period. In conclusion, Fe is involved in maintaining the period length of circadian rhythm, possibly by acting on specific central oscillators through a retrograde signaling pathway.

Exposing cells to excess metal concentrations well beyond the cellular quota is a powerful tool for understanding the molecular mechanisms of metal homeostasis. Such improved understanding may enable bioengineering of organisms with improved nutrition and bioremediation capacity. We report here that Chlamydomonas reinhardtii can accumulate manganese (Mn) in proportion to extracellular supply, up to 30-fold greater than its typical quota and with remarkable tolerance. As visualized by X-ray fluorescence microscopy and nanoscale secondary ion MS (nanoSIMS), Mn largely co-localizes with phosphorus (P) and calcium (Ca), consistent with the Mn-accumulating site being an acidic vacuole, known as the acidocalcisome. Vacuolar Mn stores are accessible reserves that can be mobilized in Mn-deficient conditions to support algal growth. We noted that Mn accumulation depends on cellular polyphosphate (polyP) content, indicated by 1) a consistent failure of C. reinhardtii vtc1 mutant strains, which are deficient in polyphosphate synthesis, to accumulate Mn and 2) a drastic reduction of the Mn storage capacity in P-deficient cells. Rather surprisingly, X-ray absorption spectroscopy, EPR, and electron nuclear double resonance revealed that only little Mn2+ is stably complexed with polyP, indicating that polyP is not the final Mn ligand. We propose that polyPs are a critical component of Mn accumulation in Chlamydomonas by driving Mn relocation from the cytosol to acidocalcisomes. Within these structures, polyP may, in turn, escort vacuolar Mn to a number of storage ligands, including phosphate and phytate, and other, yet unidentified, compounds.

Summary The direct analysis of phytosiderophores (PSs) and their metal complexes in plants is critical to understanding the biological functions of different PSs. Here we report on a rapid and highly sensitive liquid chromatography‐electrospray ionization‐quadrupole‐time of flight‐mass spectrometry (LC‐ESI‐Q‐TOF‐MS) method for the direct and simultaneous determination of free PSs and their ferric complexes in plants. In addition to previously reported PSs – deoxymugineic acid (DMA), mugineic acid (MA) and epihydroxymugineic acid (epi‐HMA) – two more PSs, avenic acid (AVA) and hydroxyavenic acid (HAVA), were identified by this method in roots of Hordeum vulgare cv Himalaya and in root exudates under iron (Fe) deficiency. The two identified PSs could be responsible for Fe acquisition under Fe deficiency because of their relative abundance and ability to form ferric complexes in secreted root exudates. This LC‐ESI‐Q‐TOF‐MS method greatly facilitates the identification of free PSs and PS–Fe complexes in one plant sample.

Hyperaccumulators tolerate and accumulate extraordinarily high concentrations of heavy metals. Content of the metal chelator nicotianamine (NA) in the root of zinc hyperaccumulator Arabidopsis halleri is elevated compared with nonhyperaccumulators, a trait that is considered to be one of the markers of a hyperaccumulator. Using metabolite-profiling analysis of root secretions, we found that excess zinc treatment induced secretion of NA in A. halleri roots compared with the nonhyperaccumulator Arabidopsis thaliana. Metal speciation analysis further revealed that the secreted NA forms a stable complex with Zn(II). Supplying NA to a nonhyperaccumulator species markedly increased plant zinc tolerance by decreasing zinc uptake. Therefore, NA secretion from A. halleri roots facilitates zinc hypertolerance through forming a Zn(II)-NA complex outside the roots to achieve a coordinated zinc uptake rate into roots. Secretion of NA was also found to be responsible for the maintenance of iron homeostasis under excess zinc. Together our results reveal root-secretion mechanisms associated with hypertolerance and hyperaccumulation.

Electrospray ionization-mass spectrometry (ESI-MS) is used to analyze metal species in a variety of samples. Here, we describe an application for identifying metal species by tandem mass spectrometry (ESI-MS/MS) with the release of free metals from the corresponding metal–ligand complexes. The MS/MS data were used to elucidate the possible fragmentation pathways of different metal–deoxymugineic acid (–DMA) and metal–nicotianamine (–NA) complexes and select the product ions with highest abundance that may be useful for quantitative multiple reaction monitoring. This method can be used for identifying different metal–ligand complexes, especially for metal species whose mass spectra peaks are clustered close together. Different metal–DMA/NA complexes were simultaneously identified under different physiological pH conditions with this method. We further demonstrated the application of the technique for different plant samples and with different MS instruments.

SummaryTo acquire appropriate iron (Fe), vascular plants have developed two unique strategies, the reduction-based strategy I of nongraminaceous plants for Fe 2+ and the chelation-based strategy II of graminaceous plants for Fe 3+. However, the mechanism of Fe uptake in bryophytes, the earliest diverging branch of land plants and dominant in gametophyte generation is less clear.Fe isotope fractionation analysis demonstrated that the liverwort Marchantia polymorpha uses reduction-based Fe acquisition. Enhanced activities of ferric chelate reductase and proton ATPase were detected under Fe-deficient conditions. However, M. polymorpha did not show mugineic acid family phytosiderophores, the key components of strategy II, or the precursor nicotianamine.Five ZIP (ZRT/IRT-like protein) homologs were identified and speculated to be involved in Fe uptake in M. polymorpha. MpZIP3 knockdown conferred reduced growth under Fe-deficient conditions, and MpZIP3 overexpression increased Fe content under excess Fe.Thus, a nonvascular liverwort, M. polymorpha, uses strategy I for Fe acquisition. This system may have been acquired in the common ancestor of land plants and coopted from the gametophyte to sporophyte generation in the evolution of land plants.