University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Agronomy & Horticulture -- Faculty Publications Agronomy and Horticulture Department 2014 Transcriptomic and physiological characterization of the fefe mutant of melon (Cucumis melo) reveals new aspects of iron-copper crosstalk Brian M. Waters University of Nebraska-Lincoln, [email protected] Samuel A. McInturf University of Nebraska-Lincoln Keenan Amundsen University of Nebraska-Lincoln, [email protected] Follow this and additional works at: https://digitalcommons.unl.edu/agronomyfacpub Part of the Agricultural Science Commons, Agriculture Commons, Agronomy and Crop Sciences Commons, Botany Commons, Horticulture Commons, Other Plant Sciences Commons, and the Plant Biology Commons Waters, Brian M.; McInturf, Samuel A.; and Amundsen, Keenan, "Transcriptomic and physiological characterization of the fefe mutant of melon (Cucumis melo) reveals new aspects of iron-copper crosstalk" (2014). Agronomy & Horticulture -- Faculty Publications. 829. https://digitalcommons.unl.edu/agronomyfacpub/829 This Article is brought to you for free and open access by the Agronomy and Horticulture Department at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Agronomy & Horticulture -- Faculty Publications by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Published in New Phytologist 203 (2014), pp. 1128–1145; doi: 10.1111/nph.12911 Copyright © 2014 Brian M. Waters, Samuel A. McInturf, and Keenan Amundsen. Used by permission. New Phytologist © 2014 New Phytologist Trust; published by John Wiley & Sons. Submitted February 3, 2014; accepted May 9, 2014 digitalcommons.unl.edudigitalcommons.unl.edu Supporting information for this article follows the references. Transcriptomic and physiological characterization of the fefe mutant of melon (Cucumis melo) reveals new aspects of iron-copper crosstalk Brian M. Waters, Samuel A. McInturf and Keenan Amundsen Department of Agronomy and Horticulture, University of Nebraska, Lincoln NE 68583-0915, USA Corresponding author – Brian M. Waters, email [email protected] Abstract Iron (Fe) and copper (Cu) homeostasis are tightly linked across biology. In previous work, Fe deficiency interacted with Cu-regu- lated 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 tran- scription factor genes. Numerous genes were regulated by both Fe and Cu. The fefe mutant was rescued by high Fe or by Cu defi- ciency, which stimulated ferric-chelate reductase activity, FRO2 expression, and Fe accumulation. Accumulation of Fe in Cu-de- ficient 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 reg- ulation of Fe uptake in plants. Keywords: copper (Cu), fefe mutant, ferricchelate reductase, iron (Fe), iron-copper crosstalk, melon (Cucumis melo), metal homeostasis Introduction and bHLH039 (Yuan et al., 2008; Wang et al., 2013), and these proteins also have regulatory roles independent of FIT (Siv- Iron (Fe) and copper (Cu) are trace metals that are required by itz et al., 2012; Wang et al., 2013). Several metal homeostasis plants for their roles in redox metabolism, such as mitochon- genes respond to both Fe and Cu, such as the metal transport- drial respiration, photosynthesis, and nitrogen fixation (Puig ers COPT2 and ZIP2, and the ferric-chelate reductase FRO3 et al., 2007; Burkhead et al., 2009; Hansch & Mendel, 2009; Pi- (Sancenon et al., 2003; Wintz et al., 2003; Colangelo & Gueri- lon et al., 2011). Excess Fe or Cu leads to oxidative stress and not, 2004; Mukherjee et al., 2006; Buckhout et al., 2009; Garcia damage from reactive oxygen species (Halliwell & Gutter- et al., 2010; del Pozo et al., 2010; Yang et al., 2010; Stein & Wa- idge, 1992). However, Fe and Cu are both involved in protec- ters, 2012; Waters et al., 2012). Similarly, Cu deficiency results tion from reactive oxygen species (Hansch & Mendel, 2009) in up-regulated ferric-chelate reductase activity in roots (Nor- as components of peroxidases, catalase, and superoxide dis- vell et al., 1993; Welch et al., 1993; Cohen et al., 1997; Romera mutases (SODs). Iron-containing SODs (FeSODs) and Cu- et al., 2003; Chen et al., 2004). Arabidopsis FRO4 and FRO5 are containing SODs (CuSODs) are functionally interchangeable, up-regulated by Cu deficiency but not by Fe deficiency (Bernal but are products of different genes (Kliebenstein et al., 1998; et al., 2012), and provide low-level but significant ferricchelate Alscher et al., 2002; Myouga et al., 2008; Pilon et al., 2011). reductase activity. Iron deficiency responses include increased expression of Changes in availability of one mineral nutrient often results certain genes to increase Fe uptake and to make cellular ad- in changes in homeostasis of other minerals. For example, Fe justments to maintain homeostasis. The basic helix-loop-helix deficiency caused changes in the expression of genes related to (bHLH) transcription factor FIT is required for normal regula- potassium and phosphate (Wang et al., 2002) and sulfate (Pao- tion of Fe-uptake genes in Arabidopsis (Colangelo & Guerinot, lacci et al., 2013) homeostasis. Fe homeostasis interacts with 2004; Jakoby et al., 2004), including the ferric-chelate reductase Zn tolerance (Pineau et al., 2012), and Cu deficiency interacts FRO2, the primary Fe transporter IRT1, and another Fe trans- with phosphate signaling (Perea-García et al., 2013) and cad- porter, NRAMP1. The FIT protein interacts with other Fe-regu- mium tolerance (Gayomba et al., 2013). Copper concentration lated bHLH proteins, such as bHLH100, bHLH101, bHLH038, was higher in Fe-deficient leaves (Welch et al., 1993; Chaignon 1128 T RANSCRIP T OMIC AND PHYSIOLOGICAL CHARAC T ERIZA T ION OF T HE FEFE MU T AN T OF MELON 1129 et al., 2002; Waters & Troupe, 2012; Waters et al., 2012). Sev- EDDHA (Sprint 138, Becker-Underwood, Ames, IA, USA), eral Cu-responsive genes and microRNAs had altered abun- 25 µM CaCl2, 25µM H3BO3, 2µM MnCl2, 2µM ZnSO4, 0.5 µM dance under Fe deficiency in Arabidopsis thaliana (Stein & Wa- CuSO4, 0.5 µM Na2MoO4 and 1 mM MES buffer (pH 5.5) or, ters, 2012; Waters et al., 2012). This suggested that a specific if indicated, HEPES buffer (pH 7.1). Fe was omitted or sup- role for accumulation of Cu under Fe deficiency is the replace- plied as indicated for Fe-supply treatments, and Cu was omit- ment of FeSOD, which decreases under Fe deficiency (Kurepa ted or supplied as indicated for Cu-supply treatments. For N et al., 1997; Waters et al., 2012), with CuSOD, whose tran- source experiments, the same micronutrients were used, with scripts increase in Fe-deficient leaves (Waters et al., 2012). Sup- a macronutrient solution as follows: 0.7mM K2SO4, 0.1mM porting this hypothesis, counteraction of oxidative stress was KH2PO4, 0.1mM KCl, 0.5mM MgSO4, and 1 mM CaCl2. Nitro- impaired when formation of functional CuSOD protein was gen was added at a final concentration of 2.5mM as (NH4)2SO4 blocked under Fe deficiency (Waters et al., 2012). Increasing or KNO3. evidence points to the importance of Fe-Cu crosstalk in metal Melon and cucumber seeds were sprouted on germina- homeostasis (Bernal et al., 2012; Waters et al., 2012; Perea-Gar- tion paper in a 30°C incubator until transplanting to hydro- cía et al., 2013). ponics after 4 d. Seedlings were placed in sponge holders in Mutant lines with altered metal homeostasis are valu- lids of black plastic containers, four plants per 750 ml solu- able tools to study molecular and physiological responses to tion. Plants were grown in a growth chamber with a mix of metal stress. The fefe mutation originated spontaneously in the incandescent and fluorescent light at 300 µmol m–2s–1. For melon (Cucumis melo) variety Edisto, and was crossed into the cucumber, plants were pretreated in standard solution for variety Mainstream to generate the C940-fe germplasm (Nu- 5 d before nutrient treatments for 3 d. For the –Cu fefe mu- gent & Bhella, 1988; Nugent, 1994). The fefe mutant lacks ferric- tant rescue and WT controls, seedlings were grown without chelate reductase activity and rhizosphere acidification (Jolley Cu from initial planting for 9 d. Plants for the +/– Cu RNA- et al., 1991), two of the important mechanisms of the reductive seq experiment (Edisto and fefe) were collected at 9 d. For the strategy of Fe uptake in dicots and nongrass monocots. The fefe +/– Fe RNA-seq experiment, WT (Edisto and snake melon) mutant has chlorotic leaves typical of Fe deficiency, which can and fefe mutants were pretreated for 9 d on –Cu solution, and be corrected by application of external Fe. These signs point to only fefe mutants that had green leaves were used for treat- fefe as a regulator of Fe uptake, but the mutant was not fully ments of 3 d duration. The purpose of the –Cu pretreatment physiologically characterized to determine if the mutation is was to use only healthy fefe plants so that the transcriptome specific to root function. Additionally, gene expression levels would reflect the Fe-regulated genes in fefe rather than sec- in fefe had not been characterized. ondary effects of severe Fe deficiency. To avoid potential Our overall objective in this study was to use the fefe mu- variation resulting from the circadian clock, sampling for fer- tant to increase understanding of Fe-uptake regulation and to ric-chelate reductase activity or RNA was always performed explore Fe-Cu crosstalk through characterization of transcrip- between 14:00 and 16:00 h.