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Copper and ectopic expression of the Arabidopsis transport protein COPT1 alter iron homeostasis in rice (Oryza sativa L.)

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Copper deficiency and excess differentially affect iron homeostasis in rice and overexpression of the Arabidopsis high-affinity copper transporter COPT1 slightly increases endogenous iron concentration in rice grains.

Abstract

Higher plants have developed sophisticated mechanisms to efficiently acquire and use micronutrients such as copper and iron. However, the molecular mechanisms underlying the interaction between both metals remain poorly understood. In the present work, we study the effects produced on iron homeostasis by a wide range of copper concentrations in the growth media and by altered copper transport in Oryza sativa plants. Gene expression profiles in rice seedlings grown under copper excess show an altered expression of genes involved in iron homeostasis compared to standard control conditions. Thus, ferritin OsFER2 and ferredoxin OsFd1 mRNAs are down-regulated whereas the transcriptional iron regulator OsIRO2 and the nicotianamine synthase OsNAS2 mRNAs rise under copper excess. As expected, the expression of OsCOPT1, which encodes a high-affinity copper transport protein, as well as other copper-deficiency markers are down-regulated by copper. Furthermore, we show that Arabidopsis COPT1 overexpression (C1 OE) in rice causes root shortening in high copper conditions and under iron deficiency. C1 OE rice plants modify the expression of the putative iron-sensing factors OsHRZ1 and OsHRZ2 and enhance the expression of OsIRO2 under copper excess, which suggests a role of copper transport in iron signaling. Importantly, the C1 OE rice plants grown on soil contain higher endogenous iron concentration than wild-type plants in both brown and white grains. Collectively, these results highlight the effects of rice copper status on iron homeostasis, which should be considered to obtain crops with optimized nutrient concentrations in edible parts.

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Acknowledgements

This work has been supported by grants BIO2011-24848 and BIO2014-56298-P from the Spanish Ministry of Economy and Competitiveness, and by FEDER funds from the European Union. We acknowledge Àngela Carrió-Seguí (Universitat de València) and Kiranmayee Pamidimukkala for their technical help with this manuscript. We also acknowledge the Servei Central d’Instrumentació Científica (Universitat Jaume I) and SCSIE (Universitat de València) for the ICP-MS and atomic absorption spectrophotometry determinations and greenhouse facilities.

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SP and LP conceived the idea and wrote the manuscript. AA-B, FA and CD perform the rice microarray and transformation experiments. AA-B, AG-M and AP-G perform the physiological and molecular experiments in rice plants.

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Correspondence to Lola Peñarrubia.

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The authors declare that they have no conflict of interest.

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11103_2017_622_MOESM1_ESM.pdf

Fig. S1 Hormone concentration in O. sativa plants. (a) ABA concentration of the 8 day-old seedlings of the WT plants grown under severe deficiency (BCS 100 µM) or sufficiency (10 µM CuSO4) of Cu. (b) JA concentration of the 8 day-old seedlings of the WT plants grown under same conditions as in (a). Represented values are the mean±SD of n=3 replicates. Samples with a common letter are not significantly different (p-value >0.05).

Fig. S2 Gene expression in the WT plants with different Cu status. (a) Relative gene expression of OsCOPT5, OsCOPT7, OsATX1 of the WT 8-day-old seedlings grown on control conditions (0 µM CuSO4) and excess (75 µM CuSO4). (b) Relative gene expression of OsNAS1, NAAT1, OsMt1f of the WT 8-day-old seedlings shoots grown in same conditions as in (a). Expression values are relative to the values of seedlings grown under Cu deficiency conditions. Represented values are the mean±SD of n=3 replicates. Samples with a common letter are not significantly different (p-value>0.05).

Fig. S3 Gene expression in the WT plants with different Cu status. (a) Relative gene expression of OsFSD1.1, OsFSD1.2, OsCDGSH of the WT 8-day-old seedlings shoots grown on a Cu scale from control conditions (0 µM CuSO4) to excess (75 µM CuSO4). (b) Relative gene expression of OsHRZ1 and OsHRZ2 of the WT 8-day-old seedlings grown in same conditions as in (a). Expression values are relative to the values of seedlings grown under Cu deficiency conditions. Represented values are the mean±SD of n=3 replicates. Samples with a common letter are not significantly different (p-value>0.05).

Fig. S4 The SOD gene expression of the WT and AtCOPT1 overexpressing rice plants. Relative expression of genes OsCSD1.1, OsCSD1.2, OsCSD2 (encoding Cu, Zn SODs), OsFSD1.1 and OsFSD1.2 (encoding Fe SODs) of the WT and C1OE 8-day-old seedling shoots grown under control conditions (0 μM CuSO4) and excess (75 μM CuSO4). Expression values are relative to the WT seedlings grown under Cu deficiency. Represented values are the mean±SD of n=3 replicates.

Fig. S5 Characterization of the WT and AtCOPT1 overexpressing rice plants grown in hydroponic cultures. (a) Representative photographs of 1 month-old plants. The WT and C1OE-1 plants were grown in different hydroponic cultures under different Cu and Fe conditions: Cu and Fe sufficiency (+Cu+Fe) and double Cu and Fe deficiency (-Cu-Fe). (b) Leaf chlorophyll content. Values of chlorophyll per gram of fresh weight of the WT and C1OE-1 1 month-old plants grown under the same conditions as in (a). Represented values are the mean±SD of n=3 replicates. Samples with a common letter are not significantly different (p-value >0.05).

Fig. S6 Metal concentration of the WT and AtCOPT1 overexpressing rice plants grown in soil. (a) Cu concentration in young and old leaves of the WT and C1OE from 4 and 5 month‐old plants grown in soil (b) Fe concentration of the WT and C1OE plants grown under the same conditions as in (a). Represented values are the mean±SD of n=3 replicates. Samples with a common letter are not significantly different (p‐value> 0.05).

Fig. S7 Perl’s staining of the brown rice grain of the WT and C1OE plants. Seeds were germinated for 3 days in ½MS and photographed after Perl’s stained.

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Andrés-Bordería, A., Andrés, F., Garcia-Molina, A. et al. Copper and ectopic expression of the Arabidopsis transport protein COPT1 alter iron homeostasis in rice (Oryza sativa L.). Plant Mol Biol 95, 17–32 (2017). https://doi.org/10.1007/s11103-017-0622-8

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  • DOI: https://doi.org/10.1007/s11103-017-0622-8

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