Transcriptomic and physiological characterization of the fefe mutant of melon (Cucumis melo) reveals new aspects of iron-copper crosstalk

Abstract

Iron (Fe) and copper (Cu) homeostasis are tightly linked across biology. In previous work, Fe deficiency interacted with Cu-regulated genes and stimulated Cu accumulation. The C940-fe (fefe) Fe-uptake mutant of melon (Cucumis melo) was characterized, and the fefe mutant was used to test whether Cu deficiency could stimulate Fe uptake. Wild-type and fefe mutant transcriptomes were determined by RNA-seq under Fe and Cu deficiency. FeFe-regulated genes included core Fe uptake, metal homeostasis, and transcription factor genes. Numerous genes were regulated by both Fe and Cu. The fefe mutant was rescued by high Fe or by Cu deficiency, which stimulated ferric-chelate reductase activity, FRO2 expression, and Fe accumulation. Accumulation of Fe in Cu-deficient plants was independent of the normal Fe-uptake system. One of the four FRO genes in the melon and cucumber (Cucumis sativus) genomes was Fe-regulated, and one was Cu-regulated. Simultaneous Fe and Cu deficiency synergistically up-regulated Fe-uptake gene expression. Overlap in Fe and Cu deficiency transcriptomes highlights the importance of Fe-Cu crosstalk in metal homeostasis. The fefe gene is not orthologous to FIT, and thus identification of this gene will provide clues to help understand regulation of Fe uptake in plants.

Keywords: copper (Cu); fefe mutant; ferric-chelate reductase; iron (Fe); iron-copper crosstalk; melon (Cucumis melo); metal homeostasis.

Fig. 1

Accumulation of iron (Fe) during seedling early growth of Cucumis melo. (a) Dry weight (DW) of wild type (WT) and fefe mutant cotyledon pairs (± SD) at planting (d0, closed bars) and after 6 d growth on complete nutrient solution (10 μM Fe, 0.5 μM Cu; open bars). (b) Fe content of cotyledon pairs (± SD) of WT and fefe mutant cotyledons at planting (d0; closed bars) and after 6 d growth (open bars) on complete solution. Significant difference between WT and fefe: *, P<0.05.

Fig. 2

The fefe mutant of Cucumis melo does not upregulate iron (Fe) uptake genes. (a) Root ferric-chelate reductase activity (± SD) after 3 d of treatment. Figure shows a representative experiment of wild type (WT) and three separate experiments of fefe roots over a range of Fe concentrations. (b) Quantitative real-time RT-PCR for FRO1, FIT, Nramp1, and IRT1 in WT and fefe mutant roots. Relative expression as normalized to ubiquitin and WT at 25 μM Fe.

Fig. 3

The fefe defect is localized to the roots of Cucumis melo as determined by grafting. (a) Shoot phenotype of grafted plants. Upper left, fefe scion grafted to fefe rootstock, upper right, fefe scion grafted to WT (Edisto) rootstock, lower left, wild type (WT) scion grafted to WT rootstock, lower right, WT scion grafted to fefe rootstock. (b) Root ferric-chelate reductase activity after transferring individual plants shown in (a) to -Fe solution for 3 d. x-axis indicates scion first and rootstock second.

Fig. 4

Copper (Cu) deficiency of Cucumis melo stimulates iron (Fe) uptake and rescues the fefe phenotype. (a) Photograph of fefe plants grown without Cu (left) and on complete nutrient solution (right). (b) Root ferric-chelate reductase activity (± SD) after transferring rescued fefe plants to -Fe-Cu or -Fe+Cu solution for 3 d. (c–e) Stacked bar graphs of (c) dry weight (DW), (d) Cu content, and (e) Fe content of plant parts for fefe and wild type (WT) plants (± SE) grown with or without Cu. Significant difference between +Cu and -Cu treatments: *, P<0.05.

Fig. 5

Iron (Fe) and copper (Cu) regulation of cucumber (Cucumis sativus) FRO genes. (a) Root ferric-chelate reductase activity (± SD) after 3 d of treatment for control (+Fe, 0.5 μM Cu) and -Fe with a range of Cu supply. (b) Quantitative real-time RT-PCR of expression (± SD) of four cucumber FRO genes from the plants in (a). Significant difference between control and treatments: *, P<0.05. (c) Phylogenetic tree drawn from ClustalW alignment of FRO protein sequences from cucumber (Cs), melon (Cm), Arabidopsis (At), Pisum sativum (Ps), and Medicago truncatula (Mt).

Fig. 6

Venn diagrams for iron (Fe) regulated genes in fefe and two wild-type (WT) Cucumis melo accessions, Edisto (Ed) and snake melon (sn). Genes of interest are shown in the appropriate set or overlap of sets. (a) Genes upregulated under Fe deficiency, (b) genes downregulated under Fe deficiency, (c) genes downregulated in fefe and upregulated in WTs, (d) genes upregulated in fefe and downregulated in WTs.

Fig. 7

Venn diagram of genes upregulated (Up) or downregulated (Dn) under copper (Cu) deficiency in fefe or wild type (WT) Edisto (Ed) Cucumis melo roots. Genes of interest are shown in the appropriate set or overlap of sets.

Fig. 8

Venn diagrams to identify number of overlapping genes in iron (Fe) and copper (Cu) regulated genes of Cucumis melo roots. (a) Genes upregulated in fefe and Edisto (Ed) under Fe deficiency and/or Cu deficiency, (b) genes downregulated in fefe and Edisto (Ed) under Fe deficiency and/or Cu deficiency. Genes of interest are shown in the appropriate set or overlap of sets.

Fig. 9

Regulation of melon (Cucumis melo) root ferric-chelate reductase activity and iron (Fe) uptake gene expression by Fe and copper (Cu). (a) Ferric-chelate reductase activity (± SD) in roots after 3 d treatment with 10 μM Fe and 0.5 μM Cu (+Fe+Cu), -Fe+Cu, +Fe-Cu, or -Fe-Cu solutions. Wild type (WT), black bars; fefe mutant, grey bars. Gene expression in roots of the plants above, for (b) FIT, (c) FRO1, (d) FRO2, (e) IRT1, (f) NRAMP1, and (g) COPT2. Significant difference between control (+Fe+Cu) and treatments: *, P<0.05.

Fig. 10

Models of effects of the fefe mutation of Cucumis melo. (a) Model of potential FIT regulation under single iron (Fe) or copper (Cu) deficiencies, or simultaneous Fe and Cu deficiency. In wild type (WT), FIT upregulates its own expression with a required partner bHLH protein, which is missing or mutated in fefe. A substitute partner protein is upregulated by Cu deficiency, which allows the fefe mutant to transcribe FIT and activate FIT targets. (b) Model of metal homeostasis alterations in fefe roots based on transcript abundance. Dashed lines represent lower expression relative to WT, solid lines represent higher expression relative to WT. Transport of Fe into a generic cell is represented for Fro1, Irt1, Nramp1, and Opt3 proteins, transport of Fe into the vacuole is represented by Vit1 and NodL proteins, transport out of a vacuole is represented by Nramp3 protein, and cytoplasmic synthesis of nicotianamine is represented by Nas protein.