| Iron is a essential micronutrient in its role as metal cofactor for many enzymes and redox reactions. Considering that Fe is among the most abundant elements in the earth's crust, it is surprising that Fe deficiency is the most common nutritional deficiency in the world's population. The poor bioavailability of Fe due to the low solubility of ferric [Fe(â…¢)] oxides, in particular in alkaline environments, is likely to account for this paradox. However, Fe is an essential On the one hand, dicots and non-graminaceous monocots use strategy â… (or reduction strategy) where the soil is acidified by H+-ATPases to solubilize iron, Fe(â…¢) is reduced by ferric chelate reductases, and Fe(â…¡) uptake is mediated by IRT1, a protein of the ZIP family. On the other hand, grasses use strategy II (or chelation strategy) where phytosiderophores [i.e. high-affinity Fe(â…¢) chelators] are released, and Fe(â…¢) chelates are subsequently taken up by the YS1transporter, a member of the OPT family. The chelation strategy is more efficient than the reduction strategy and allows grasses to survive on soils with very low iron availability. In addition, it has been shown that in rice (a strategy II plant) iron can also be taken up by an IRT1-like protein, allowing rice to directly absorb Fe(II) which is more available in submerged conditions. These observations suggest a more general role of ZIP proteins in plant Fe uptake.Organisms have therefore developed efficient homeostatic mechanisms for Fe assimilation and distribution within cells and organs. In photosynthetic organisms, Fe deficiency induces metabolic adaptations that allow the maintenance of both photosynthetic and respiratory functions. Organisms possess high-and low-affinity systems for the uptake of Fe depending on its extracellular concentration and chemical state (oxidized/reduced, free or chelated, etc.). High-affinity systems are usually highly selective for Fe and operate in Fe-deficient conditions, while low-affinity systems are less selective and are active in Fe-replete conditions. Plants have developed two strategies for high-affinity Fe uptake.Hydrogen ion concentration (pH) is an important property of soils because it affects the growth of plant roots and soil microorganisms. Root growth is generally favored in slightly acidic soils, at pH values between5.5and6.5. Soil pH determines the availability of soil nutrients. Acidity promotes the weathering of rocks releasing Fe2+and other cations include metal ions and increase the solubility of carbonates, sulfates, and phosphates, etc. Increasing the solubility of nutrients facilitates their availability.In a phenotypic screen of a activation tagged mutant collection in Arabidopsis based on a chemical-inducible XVE system, a gain-of-function mutant Doi2(doi-D;"D" denotes the dominant effect) was identified with the remarkable feature of having lower temperature in leaves on thermal imaging of drought-stressed plants than normal WT plants, and more sensitive to iron-deficiency. Molecular characterization of the mutant revealed that OLexA-46promoter fused immediately upstream of the putative translation initiation codon of At1g70690gene, a putative receptor-like kinase (designed RLK), that was overexpressed in the doi2-D mutant. To verify the identity of the DOI2gene(s), cDNA fragments encoding RLK was cloned into super promoter (SP) vector, and the resulting constructs were used to transform WT plants. The transformants displayed the typical doi2-D phenotype to various degrees of severity. These indicate that the OLexA-46promoter tagged RLK in the mutant genome represents DOI2.We investigated whether DOI2is invovled in nutrient deficient responses, doi2seeds were germinated on Murashige and Skoog medium with or without the inducer. When germinated on the noninductive medium, no apparent growth and development abnormality was observed, suggesting that mutations in the DOI2genome did not appear to affect the normal function of the gene. On the inductive medium lacking iron, however, the leaves of doi2showed severe chlorosis. Under conditions of extreme or prolonged deficiency, the veins of doi2overexpression lines also become chlorotic, causing the whole leaf to turn white. We measured the chlorophyll content at10d after germination in the nutrient medium without iron. Chlorophyll of doi2-OE leaves was19%of that in the wild type.Interestingly, DOI2gain-of-function mutation lost the soil pH regulation in the area adjacent to the root surfaces. The primary pH of MS medium near to roots of wild type and doi2gain-of function mutant was estimated using the pH indicator, bromocresol purple (BCP). When12-d-old doi2-OE and wild type were transferred to MS medium containing BCP and grown for8days, there is no difference for the pH of the medium between doi2-OE and wild type plants, in which the pH is about6.2. However, after addition of estradiol for3h, the pH of MS medium in wild type shows yellow color. The results show that the medium pH in wild type roots was about5.5, which is suitable to absorption of nutrients. However, the pH value in doi2gain-of function mutants is near6.2, which is substantially higher than that in the wild type. This means that overexprssion of DOI2function prevents the acidification medium near to roots.We were interested in the potential role of DOI2in modulating the soil pH near to plant roots. We next analyzed the effects of different medium pH values on the plant growth and iron deficiency symptoms. The seeds of wild type and the doi2-OE plants were sown on nutrient media with variation of pH under iron deficiency. the data suggest that DOI2is especially important for modulation of soil pH, and availability of nutrient in the soils.In addition, Fe deficiency induced a rapid increase of FCR activity in wild type. Compared to that of the wild-type, FCR activites of doi2mutant plants was also increased to a greater extent by a Fe deficiency. By contrast, FCR activity was inhibited in doi2gain-of-function mutants. In comparison, the reductase activity in wild type is3.5time of doi overespression mutants. This suggest that overexpression of doi2suppress the FCR activity in root surface.Fe absorption from the rhizosphere involves the enhancement of H+efflux at the root in a way that appears to induce the plasma membrane H+-ATPase in root cells. Having established that DOI2overexpression are not able to acidify the rhizosphere in the above genetic analysis, we then asked whether DOI2would interact with PM H+-ATPase. In this study, the yeast two-hybrid system was employed. Significantly, DOI2interacted strongly with DUF26-1and relatively weakly with other domains by twohybride system.In summary, we here demonstrated that the DOI2function in Fe deficiency signal transduction is closely coupled with the activity of H+-ATPase, which directly influence Fe content in Arabidopsis. Thus DOI2might sense and transduce the Fe deficiency signal to downstream components.
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