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.

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.

Overexpression of Arabidopsis ATX1 retards plant growth under severe copper deficiency.

In a previous study, we demonstrated that Arabidopsis Antioxidant Protein1 (ATX1) plays an essential role in copper (Cu) homeostasis, conferring tolerance to both excess and subclinically deficient Cu. The Cu-binding motif MXCXXC was required for the physiological function of ATX1. In this study, we found that overexpression of ATX1 resulted in hypersensitivity to severe Cu deficiency despite enhancing tolerance to subclinical Cu deficiency. However, overexpression of mutated ATX1, replacing the Cu-binding motif MXCXXC with MXGXXG, abolished the hypersensitivity, for no differences from the wild type under the same conditions. Thus, the expression of ATX1 must be cautiously regulated to avoid homeostatic imbalance with the over-chelation of Cu.

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.

Ectopic ferredoxin I protein promotes root hair growth through induction of reactive oxygen species in Arabidopsis thaliana.

Ferredoxin I (Fd-1) is a protein existing in green tissues as an electron carrier for photosynthesis. Reactive oxygen species (ROS) are generated from an over-accumulation of electrons in photosynthetic electron chains. In previous studies, plant ferredoxin-like protein (PFLP) transgenic plants could be made resistant to virulent pathogens, by inducing the generation of ROS. The generation of ROS is closely associated with root hair development, increasing with the elongation of root hairs. We propose that an ectopic expression of pflp may alter root hair development through the enhanced generation of ROS. In this report, Arabidopsis transformed with pflp was generated to determine the potential role of PFLP in root development. Transgenic Arabidopsis exhibited longer root hairs with a significant increase in endogenous H(2)O(2) compared with wild type. The growth of transgenic lines in root hairs was inhibited when treated with NADPH oxidase inhibitor. Results suggest that an over-expression of pflp had enhanced the accumulation of H(2)O(2) in the roots and further promoted the growth of root hairs. Transcriptional activities of root hair development-related and redox-regulated genes were mediated through increased levels of ROS, to alter the growth of transgenic lines in root hairs. In summary, we propose that an ectopic expression of pflp promotes root hair growth, resulting from an enhancement of ROS production.