The Function Of Iron Chelate Reductase In Iron Metabolism Of Plants

Iron is the essential micro-nutritional element for plant growth and development, which plays an important role in the physiological mechanism. Although iron is abundant in the crust, its bioavailability in soil is very low. Iron deficiency will cause the crop yield and quality, so iron deficiency chlorosis of plants has become a worldwide problem of malnutrition. By studying the physiological and molecular mechanisms of activation, absorption, metabolism and accumulation of the plant for iron in the soil, we can improve iron absorption and accumulation mode with modern biological methods, cultivate new varieties of crops of efficient iron, increase crop yields, and improve human iron nutritional status. To improve the iron content of plants, our laboratory had transformed soybean ferritin gene into the tobacco chloroplast genome, and the expression of ferritin transformants amounts to 15% of the total soluble protein; but the iron concentration in the transgenic plants is only 2-3fold higher than in the control plants, and the transformants had phenotypes of iron deficiency chlorosis.To study further iron metabolism in the Strategy I, we have over- expressed the iron chelate reductase AtFRO6 and AtFRO2 in wild-type tobacco and ferritin transplastomic tobacco. By analyzing the changes of physiological functions of transgenic plants, we further clarify the function of iron chelate reductase in iron metabolism of plants, and deepen our understanding of the molecular mechanism of iron metabolism in plants. The main results are as follows:1. The cDNA of Arabidopsis FRO6 and FRO2 gene were cloned by RT-PCR. The plant expression vector pBI121FRO6 and the plant expression vector pCAMFRO2FRO6 were constructed. The expression of the gene AtFRO6 was regulated by the CaMV35S promoter, and the expression of the gene FRO6 and FRO2 were regulated by the CaMV35S promoter.2. The FRO6 was transformed into the nuclear genome of wild-type tobacco and ferritin transplastomic tobacco by Agrobacterium mediated method. 18 strains of wild-type tobacco transformed by FRO6(6WT) and 18 strains of ferritin transplastomic tobacco transformed by FRO6(6Fe) were identified by PCR, Southern blot detection and Northern blot detection.3. The FRO6 and FRO2 were transformed into the nuclear genome of wild-type tobacco and ferritin transplastomic tobacco by Agrobacterium mediated method. 14 strains of wild-type tobacco transformed by FRO6 and FRO2 (8WT) and 15 strains of ferritin transplastomic tobacco transformed by FRO6 and FRO2 (8Fe) were identified by PCR, Southern blot detection and Northern blot detection.4. The leaf ferric reductase activity, the ferrous iron content in the leaves and the chlorophyll concentration of the transgenic plants (6WT and 6Fe) grown under iron-sufficient or iron-deficient conditions were higher respectively than that of the control plants. The chlorophyll concentration in the transgenic plants (6WT and 6Fe) grown on MS medium lacking sucrose and containing 100μM Fe (III)-EDTA was higher than control plants. The root reductase activity, the ferrous iron content in the roots, the total iron concentration in the leaves and in the roots of the transgenic plants showed no significant difference when compared to the control plants in both iron deficient and iron sufficient conditions.5. The leaf ferric reductase activity, the ferrous iron content in the leaves and the chlorophyll concentration of the transgenic plants (8WT and 8Fe) grown under iron-sufficient or iron-deficient conditions were higher respectively than that of the control plants. The root ferric reductase activity, the ferrous iron content in the roots, the total iron concentration in the roots and leaves of the transgenic plants grown under iron-deficient conditions were higher respectively than that of the control plants. The root reductase activity, the ferrous iron content in the roots, the total iron concentration in the leaves and in the roots of the transgenic plants showed no significant difference when compared to the control plants in iron sufficient conditions. The chlorophyll concentration in the transgenic plants (8WT and 8Fe) grown on MS medium lacking sucrose and containing 100μM Fe (III)-EDTA was higher than control plants.6. Our study shows that the AtFRO6 functions as a ferric chelate reductase for iron uptake by leaf cells. These results suggest that the overexpression of FRO6 in transgenic tobacco plants enhances ferric reductase activity in leaves and photosynthesis, leads to increased concentrations of ferrous iron and chlorophyll, improves leaf cells' ability of absorbing iron and plant iron nutritional status, and reduces iron deficiency chlorosis compared to control plants, and improves plant ability for low-iron resistance. These results indicate that AtFRO6 is not induced by iron deficiency and the expression of AtFRO6 may be controlled at the posttranscriptional level. 7. The Physiological data of tobacco transformed by FRO2 and FRO6 indicates that ferric reductase activity in leaves and in roots of transgenic plants was higher respectively than that of the control plants under iron-deficient conditions, but the single ferric reductase activity in leaves of tobacco transformed by FRO6 was higher respectively than that of the control plants under iron-deficient conditions and the amount was less higher than the combination with transferred FRO2 and FRO6. So FRO2 and FRO6 multi-gene transformation is important for improving resistant to iron deficiency of crops.8. Yellow phenotype of ferritin transplastomic tobacco transformed FRO6 or FRO6 and FRO2 was improved to some extent. We speculate that ferritin overaccumulation in chloroplast of ferritin transplastomic tobacco leads to an illegitimate iron sequestration and disturbs the iron homeostasis in chloroplast, resulting in reduction of free iron for physiological responses of plants, as revealed by leaf yellowing. The transgenic plant 6Fe and 8Fe alleviated this change in balance of iron homeostasis to some extent because the overexpression of FRO6 in transgenic plants enhances leaf cells' ability of absorbing iron and the overexpression of FRO6 and FRO2 enhances leaf cells' and root' ability of absorbing iron.