Exogenous Copper Application for the Elemental Defense of Rice Plants against Rice Leaffolder (Cnaphalocrocis medinalis).

Metals that accumulate in plants may confer protection against herbivorous insects, a phenomenon known as elemental defense. However, this strategy has not been widely explored in important crops such as rice (Oryza sativa L.), where it could help to reduce the use of chemical pesticides. Here, we investigated the potential of copper (Cu) and iron (Fe) micronutrient supplements for the protection of rice against a major insect pest, the rice leaffolder (Cnaphalocrocis medinalis). We found that intermediate levels of Cu (20 µM CuSO4) and high concentrations of Fe (742 µM Fe) did not inhibit the growth of C. medinalis larvae but did inhibit rice root growth and reduce grain yield at the reproductive stage. In contrast, high levels of Cu (80 µM CuSO4) inhibited C. medinalis larval growth and pupal development but also adversely affected rice growth at the vegetative stage. Interestingly, treatment with 10 µM CuSO4 had no adverse effects on rice growth or yield components at the reproductive stage. These data suggest that pest management based on the application of Cu may be possible, which would be achieved by a higher effective pesticide dose to prevent or minimize its phytotoxicity effects in plants.

Insight into the mechanism of indium toxicity in rice.

Indium is widely used in the technology industry and is an emerging form of environmental pollution. The presence of indium in soil and groundwater inhibits shoot and root growth in crops, thus reducing yields. However, the underlying mechanisms are unknown, making it difficult to design effective countermeasures. We explored the spatiotemporal effects of excess indium on the morphological, physiological and biochemical properties of rice (Oryza sativa L.). Indium accumulated mainly in the roots, severely restricting their growth and causing the acute perturbation of phosphorus, magnesium and iron homeostasis. Other effects included leaf necrosis and anatomical changes in the roots (thinned sclerenchyma and enlarged epidermal and exodermal layers). Whole-transcriptome sequencing revealed that rice immediately responded to indium stress by activating genes involved in heavy metal tolerance and phosphate starvation responses, including the expression of genes encoding phosphate-regulated transcription factors and transporters in the roots. Direct indium toxicity rather than phosphate deficiency was identified as the major factor affecting the growth of rice plants, resulting in the profound phenotypic changes we observed. The application of exogenous phosphate alleviated indium toxicity by reducing indium uptake. Our results suggest that indium immobilization could be used to prevent indium toxicity in the field.

Divalent nutrient cations: Friend and foe during zinc stress in rice.

Zn deficiency is the most common micronutrient deficit in rice but Zn is also a widespread industrial pollutant. Zn deficiency responses in rice are well documented, but comparative responses to Zn deficiency and excess have not been reported. Therefore, we compared the physiological, transcriptional and biochemical properties of rice subjected to Zn starvation or excess at early and later treatment stages. Both forms of Zn stress inhibited root and shoot growth. Gene ontology analysis of differentially expressed genes highlighted the overrepresentation of Zn transport and antioxidative defense for both Zn stresses, whereas diterpene biosynthesis was solely induced by excess Zn. Divalent cations (Fe, Cu, Ca, Mn and Mg) accumulated in Zn-deficient shoots but Mg and Mn were depleted in the Zn excess shoots, mirroring the gene expression of non-specific Zn transporters and chelators. Ascorbate peroxidase activity was induced after 14 days of Zn starvation, scavenging H2 O2 more effectively to prevent leaf chlorosis via the Fe-dependent Fenton reaction. Conversely, excess Zn triggered the expression of genes encoding Mg/Mn-binding proteins (OsCPS2/4 and OsKSL4/7) required for antimicrobial diterpenoid biosynthesis. Our study reveals the potential role of divalent cations in the shoot, driving the unique responses of rice to each form of Zn stress.

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.

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.

Evolutionary analysis of iron (Fe) acquisition system in Marchantia polymorpha.

To 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.

IRT1 degradation factor1, a ring E3 ubiquitin ligase, regulates the degradation of iron-regulated transporter1 in Arabidopsis.

Fe is an essential micronutrient for plant growth and development; plants have developed sophisticated strategies to acquire ferric Fe from the soil. Nongraminaceous plants acquire Fe by a reduction-based mechanism, and graminaceous plants use a chelation-based mechanism. In Arabidopsis thaliana, which uses the reduction-based method, iron-regulated transporter1 (IRT1) functions as the most important transporter for ferrous Fe uptake. Rapid and constitutive degradation of IRT1 allows plants to quickly respond to changing conditions to maintain Fe homeostasis. IRT1 degradation involves ubiquitination. To identify the specific E3 ubiquitin ligases involved in IRT1 degradation, we screened a set of insertional mutants in RING-type E3 ligases and identified a mutant that showed delayed degradation of IRT1 and loss of IRT1-ubiquitin complexes. The corresponding gene was designated IRT1 degradation factor1 (IDF1). Evidence of direct interaction between IDF1 and IRT1 in the plasma membrane supported the role of IDF1 in IRT1 degradation. IRT1 accumulation was reduced when coexpressed with IDF1 in yeast or Xenopus laevis oocytes. IDF1 function was RING domain dependent. The idf1 mutants showed increased tolerance to Fe deficiency, resulting from increased IRT1 levels. This evidence indicates that IDF1 directly regulates IRT1 degradation through its RING-type E3 ligase activity.

Control of Zn uptake in Arabidopsis halleri: a balance between Zn and Fe.

Zinc (Zn) is an essential plant micronutrient but is toxic in excess. To cope with excess Zn, plant species possess a strict metal homeostasis mechanism. The Zn hyperaccumulator Arabidopsis halleri has developed various adaptive mechanisms involving uptake, chelation, translocation and sequestration of Zn. In this mini review, we broadly discuss the different Zn tolerance mechanisms and then focus on controlled Zn uptake in A. halleri. Members of the ZRT/IRT-like protein (ZIP) family of metal transporters are mainly regulated by Zn and are involved in Zn uptake. A few members of the ZIP family, such as IRT1 and IRT2, are regulated by iron (Fe) and can transport multi-metals, including Zn, Fe, Mn, Cd, and Co. This mini-review also discusses the differential expression of multiple metal ZIP transporters in A. halleri and A. thaliana, a non-hyperaccumulator, with Zn exposure as well as Fe deficiency and their role in controlled Zn uptake and tolerance.

Iron is involved in the maintenance of circadian period length 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.

Copper chaperone antioxidant protein1 is essential for copper homeostasis.

Copper (Cu) is essential for plant growth but toxic in excess. Specific molecular mechanisms maintain Cu homeostasis to facilitate its use and avoid the toxicity. Cu chaperones, proteins containing a Cu-binding domain(s), are thought to assist Cu intracellular homeostasis by their Cu-chelating ability. In Arabidopsis (Arabidopsis thaliana), two Cu chaperones, Antioxidant Protein1 (ATX1) and ATX1-Like Copper Chaperone (CCH), share high sequence homology. Previously, their Cu-binding capabilities were demonstrated and interacting molecules were identified. To understand the physiological functions of these two chaperones, we characterized the phenotype of atx1 and cch mutants and the cchatx1 double mutant in Arabidopsis. The shoot and root growth of atx1 and cchatx1 but not cch was specifically hypersensitive to excess Cu but not excess iron, zinc, or cadmium. The activities of antioxidant enzymes in atx1 and cchatx1 were markedly regulated in response to excess Cu, which confirms the phenotype of Cu hypersensitivity. Interestingly, atx1 and cchatx1 were sensitive to Cu deficiency. Overexpression of ATX1 not only enhanced Cu tolerance and accumulation in excess Cu conditions but also tolerance to Cu deficiency. In addition, the Cu-binding motif MXCXXC of ATX1 was required for these physiological functions. ATX1 was previously proposed to be involved in Cu homeostasis by its Cu-binding activity and interaction with the Cu transporter Heavy metal-transporting P-type ATPase5. In this study, we demonstrate that ATX1 plays an essential role in Cu homeostasis in conferring tolerance to excess Cu and Cu deficiency. The possible mechanism is discussed.

Differential expression and regulation of iron-regulated metal transporters in Arabidopsis halleri and Arabidopsis thaliana--the role in zinc tolerance.

To avoid zinc (Zn) toxicity, plants have developed a Zn homeostasis mechanism to cope with Zn excess in the surrounding soil. In this report, we uncovered the difference of a cross-homeostasis system between iron (Fe) and Zn in dealing with Zn excess in the Zn hyperaccumulator Arabidopsis halleri ssp. gemmifera and nonhyperaccumulator Arabidopsis thaliana. Arabidopsis halleri shows low expression of the Fe acquisition and deficiency response-related genes IRT1 and IRT2 compared with A. thaliana. In A. thaliana, lowering the expression of IRT1 and IRT2 through the addition of excess Fe to the medium increases Zn tolerance. Excess Zn induces significant Fe deficiency in A. thaliana and reduces Fe accumulation in shoots. By contrast, the accumulation of Fe in shoots of A. halleri was stable under various Zn treatments. Root ferric chelate reductase (FRO) activity and expression of FIT are low in A. halleri compared with A. thaliana. Overexpressing a ZIP family member IRT3 in irt1-1, rescues the Fe-deficient phenotype. A fine-tuned Fe homeostasis mechanism in A. halleri maintains optimum Fe level by Zn-regulated ZIP transporters and prevents high Zn uptake through Fe-regulated metal transporters, and in part be responsible for Zn tolerance.

Genes associated with heavy metal tolerance and accumulation in Zn/Cd hyperaccumulator Arabidopsis halleri: a genomic survey with cDNA microarray.

To survive in variable soil conditions, plants possess homeostatic mechanisms to maintain a suitable concentration of essential heavy metal ions. Certain plants, inhabiting heavy metal-enriched or -contaminated soil, thus are named hyperaccumulators. Studying hyperaccumulators has great potential to provide information for phytoremediation. To better understand the hyperaccumulating mechanism, we used an Arabidopsis cDNA microarray to compare the gene expression of the Zn/Cd hyperaccumulator Arabidopsis halleri and a nonhyperaccumulator, Arabidopsis thaliana. By analyzing the expression of metal-chelators, antioxidation-related genes, and transporters, we revealed a few novel molecular features. We found that metallothionein 2b and 3, APX and MDAR4 in the ascorbate-glutathione pathway, and certain metal transporters in P(1B)-type ATPase, ZIP, Nramp, and CDF families, are expressed at higher levels in A. halleri than in A. thaliana. We further validated that the enzymatic activity of ascorbate peroxidase and class III peroxidases are highly elevated in A. halleri. This observation positively correlates with the higher ability of A. halleri to detoxify H2O2 produced by cadmium and paraquat treatments. We thus suggest that higher peroxidase activities contribute to the heavy metal tolerance in A. halleri by alleviating the ROS damage. We have revealed genes that could be candidates for the future engineering of plants with large biomass for use in phytoremediation.