Iron is an essential mineral nutrient for plants (Guerinot and Yi, 1994; Briat et al., 2015). It serves as a metal cofactor required for hundreds of metabolic enzymes in the energy-yielding electron transfer reactions of respiration and photosynthesis (Briat et al., 2007; Balk and Schaedler, 2014). Perturbations in iron homeostasis can lead to cytotoxicity in the plant cell, reduction of growth and organ development, and eventually chlorosis and reduced crop yield (Connolly and Guerinot, 2002; Briat et al., 2007). Therefore, iron sensing and uptake from the soil, translocation within the plant, and intracellular storage and trafficking are tightly regulated in plants (Curie and Briat, 2003; Hell and Stephan, 2003; Jeong and Guerinot, 2009; Conte and Walker, 2011; Kobayashi and Nishizawa, 2012).

The iron deficiency response is thought to be controlled by a complex regulatory network involving multiple signaling pathways and its interplay with hormones (Hindt and Guerinot, 2012; Xia et al., 2015). Early studies indicate that adaptation to iron deficiency requires remodeling of the photosynthetic apparatus to minimize the photooxidative damage caused by reactive oxygen species (ROS) (Moseley et al., 2002). Genome-wide analyses of both transcript and protein expression profiles reveal significant changes in the expression of genes and/or proteins involved in antioxidant and oxidative stress response pathways (O’Rourke et al., 2007; Forner-Giner et al., 2010; Urzica et al., 2012; Zamboni et al., 2012; Lopez-Millan et al., 2013). In particular, expression of genes encoding glutaredoxins (Grxs) and thioredoxins (Trxs) is significantly enhanced under iron deficiency (Urzica et al., 2012). Furthermore, hydrogen peroxide (H₂O₂) production is increased in roots of plants grown under iron deficiency (Le et al., 2016). Nonetheless, the role of ROS and the function of redox-regulatory proteins in iron deficiency response regulation has not been well defined.

Grx and Trx enzyme systems help to control cellular redox potential in plants (Meyer et al., 2009). Grxs are ubiquitous small heat-stable disulfide oxidoreductases conserved in both prokaryotes and eukaryotes (Lillig et al., 2008) and are important in redox regulation and stress response (Sanchez-Riego et al., 2013; Considine and Foyer, 2014). There is growing evidence that plant Grxs have diverse functions in transcriptional regulation of defense responses and flower development (Xing et al., 2005; Ndamukong et al., 2007; Werner and Schmülling, 2009; La Camera et al., 2011), antioxidative stress (Cheng et al., 2006; Cheng, 2008; Laporte et al., 2012), redox signaling (Zaffagnini et al., 2012), hormonal regulation and environmental adaptation (Sundaram and Rathinasabapathi, 2010; Cheng et al., 2011). Monothiol Grxs are first identified in yeast (ScGrx3, -4, and -5) and bacteria (Grx4) that have a single cysteine residue in the putative active motif (Rodriguez-Manzaneque et al., 1999; Fernandes et al., 2005). This group of Grxs is conserved across species and accumulating evidence suggests they play a unique function in regulating iron homeostasis (Herrero and de la Torre-Ruiz, 2007; Lillig et al., 2008; Rouhier et al., 2010; Stroher and Millar, 2012). Yeast ScGrx5 encodes a mitochondrial monothiol Grx, which is required for biogenesis of iron-sulfur clusters (Rodriguez-Manzaneque et al., 2002; Uzarska et al., 2013), whereas ScGrx3 and ScGrx4, through interactions with iron-regulatory transcription factors, like Aft1 and Aft2, and coactivators like Fra/BolA proteins, globally modulate iron uptake, intracellular sensing, and trafficking in yeast cells (Ojeda et al., 2006; Pujol-Carrion et al., 2006; Kumanovics et al., 2008; Mercier and Labbé, 2009; Muhlenhoff et al., 2010; Li et al., 2011; Li and Outten, 2012; Vachon et al., 2012; Tamayo et al., 2016). In plants, Arabidopsis and Poplar monothiol Grxs, such as AtGRXS14 (AtGRXcp), AtGRXS15 (AtGRX4), AtGRXS16 and AtGRXS17, bind a Fe-S cluster and are able to complement yeast ScGrx5 function in Fe-S cluster assembly when expressed in yeast mutant cells (Cheng et al., 2006; Bandyopadhyay et al., 2008; Cheng, 2008; Li et al., 2010; Liu et al., 2013; Knuesting et al., 2015). However, the function of plant monothiol Grxs in iron regulation and stress responses in planta remains to be explored.

Arabidopsis AtGRXS17 is one of four “CGFS” type monothiol Grxs in Arabidopsis (Lemaire, 2004) with one Trx-like domain at its N-terminal region and three “CGFS” containing Grx domains at its C-terminus (Cheng et al., 2006; Herrero and de la Torre-Ruiz, 2007). Our previous studies indicate that AtGRXS17 is essential for post-embryonic growth and hormonal responses in plants under elevated temperature (Cheng et al., 2011). Meanwhile, ectopic expression of AtGRXS17 enhances stress tolerance (Wu et al., 2012; Hu et al., 2015). AtGRXS17 interacts with plant BolA proteins in an in vitro study (Couturier et al., 2014) and AtGRXS17 appears to be able to bind an iron-sulfur (Fe-S) cluster (Knuesting et al., 2015), suggesting that AtGRXS17 plays an important role in iron homeostasis. In the present report, we utilize yeast expression studies and reverse genetics in Arabidopsis to study the function of AtGRXS17 under iron deficiency stress. Our findings demonstrate that AtGRXS17 plays an important role in protecting plants from iron deficiency induced oxidative damage.

Our genetic studies presented here offer insight into the relationship between redox regulation and iron homeostasis in plants. This work provides evidence that AtGRXS17 is involved in regulation of iron homeostasis in plants and helps to alleviate iron deficiency stress through mediating redox balance.

In yeast mutant cells, AtGRXS17 suppressed iron accumulation phenotypes (Figure 1). Interestingly, the suppression of iron accumulation in double mutant cells by AtGRXS17 appears to be stronger than ScGrx3 alone (Figure 1). This observation could be due to the fact that ScGrx3 and ScGrx4 functions are not completely overlapping. It has been shown that cytosolic and nuclear ScGrx3/ScGrx4 have distinct functions in iron regulation and homeostasis (Ojeda et al., 2006; Pujol-Carrion et al., 2006; Muhlenhoff et al., 2010). When ectopically expressed in yeast, AtGRXS17 is found both in the nucleus and the cytoplasm (Wu et al., 2012). It is possible that AtGRXS17 could rescue the yeast grx3grx4 phenotype better due to its dual cellular localization in yeast cells.

In yeast, deletion of both ScGrx3 and ScGrx4 results in growth defects and enhanced sensitivity to oxidative stress caused by iron accumulation (Pujol-Carrion et al., 2006; Wu et al., 2012). Similarly, AtGRXS17 loss-of-function plants displayed root and vegetative growth retardation under normal conditions (Figures 2A,C,D) (Cheng et al., 2011; Knuesting et al., 2015) and increased sensitivity to oxidative stress (Figure 4). This finding indicates that AtGRXS17 and its yeast orthologs have conserved functions. However, there is functional divergene between the plant and yeast genes as yeast grx3grx4 cells use an iron chelator to alleviate oxidative stress (Pujol-Carrion et al., 2006). Meanwhile AtGRXS17 loss-of-function seedlings were hypersensitive to iron deficiency (in the presence of iron chelator) (Figure 2).

Root iron reductase activity was significantly induced in wild type controls and mutants under iron deplete medium compared to that under iron sufficient medium (Figure 3A). It appears that atgrxs17 KO and AtGRXS17 RNAi seedlings has higher root iron reductase activities than wild type controls under both iron sufficient and deficient conditions (Figure 3A). The increase in root iron reductase activity, especially under iron sufficient medium, may not result in increased iron uptake and accumulation in mutant plants. In agreement with this, no difference in iron accumulation in mature leaves between AtGRXS17 loss-of-function plants and wild type controls was observed when plants were grown in soil. However, iron concentration in mature seeds of mutant plants was slightly, but significantly increased compared to wild type controls (Figure 3B). This suggests that AtGRXS17 may modulate iron distribution within a plant. It is also possible that a reduction in seed yield of these mutant plants could have contributed to the elevated seed iron levels through a concentrating process (i.e., the same total partitioning of iron to seeds, but to a smaller pool of seeds). Higher concentrations of calcium, magnesium, phosphorus, and zinc were also seen in seeds of soil-grown mutant plants (Figures 3C,E–G). Unfortunately, seed yield was not measured in the current study. Previous research has shown that seeds comprise about 30% of whole-shoot iron content and about 15% of shoot mass at maturity in Arabidopsis (Waters and Grusak, 2008). Thus, even a moderate lowering of seed production in the mutants could explain a portion of the increased seed iron concentration in these plants. Previous studies have shown that AtGRXS17 loss-of-function plants display subtle growth defects (Cheng et al., 2011; Knuesting et al., 2015). Furthermore, when seedlings were grown on iron sufficient medium, the growth of AtGRXS17 mutant primary roots was slower than that of wild type controls (Figures 2A,C, 6A,E). Whether those growth defects are attributed to altered iron accumulation/distribution is not clear in the current study. We posit that the increased ROS production/oxidative stress (Figures 5A,C) are the causal factors for inhibition of root growth.

Iron deficiency causes cellular oxidative stress and induces antioxidant defense genes (pathways) including Grxs in plants and green algae (Thimm et al., 2001; Connolly and Guerinot, 2002; O’Rourke et al., 2007; Lan et al., 2011; Urzica et al., 2012; Lopez-Millan et al., 2013; Le et al., 2016). Reduced GSH contents are significantly decreased in Arabidopsis plants under iron deficiency, whereas ROS levels are drastically increased (Ramirez et al., 2013). Furthermore, addition of reduced GSH can alleviate the detrimental effects of iron deficiency through controlling ROS production/oxidative stress caused by iron deficiency (Ramirez et al., 2013). Our data revealed an increase of AtGRXS17 expression (both mRNA and protein levels) in plants under iron deficiency (Supplementary Figure S1), while disruption of AtGRXS17 significantly inhibited plant growth under the same condition (Figure 2). This finding demonstrates that AtGRXS17 is required for plant survival under iron deficiency stress. Our studies support the notion that AtGRXS17 may alleviate the iron deficiency stress through mediating redox balance. First, atgrxs17 KO and AtGRXS17 RNAi seedlings are sensitive to oxidative stress (Figure 4); Second, ROS production is significantly increased in mutant roots under iron deficiency (Figures 5B,C), which is a contributing factor to cause cell damage and impair plant growth. Third, although the intracellular GSH levels in both mutant seedlings and wild type controls were not measured in the current study, addition of reduced GSH is able to suppress, at least in part, the growth defects of mutant seedlings under iron deficiency (Figure 6). Furthermore, overexpression of AtGRXS17 enhances antioxidant enzymatic activities in transgenic tomato plants (Wu et al., 2012). Taken together, these results indicate that AtGRXS17 is crucial for protecting plants from iron deficiency induced oxidative damage.

AtGRXS17, similar to ScGrx3/ScGrx4, is a Fe-S cluster binding protein (Knuesting et al., 2015) and is postulated to mediate iron or iron-sulfur cluster transfer processes (Philpott, 2012; Inigo et al., 2016). Disruption of both ScGrx3 and ScGrx4 in yeast drastically alters iron sensing, intracellular trafficking, and mitochondrial iron distribution through their bound iron-sulfur clusters (Muhlenhoff et al., 2010). Whether AtGRXS17 modulates iron deficiency responses through its bound cluster is yet to be determined. Interestingly, Arabidopsis BolA protein, an interacting partner of AtGRXS17, might play a role in iron metabolism and redox regulation independent of its iron-sulfur binding ability (Qin et al., 2015). We envision AtGRXS17 playing a myriad of roles in planta; however, studies directed at clarifying other AtGRXS17 functions require additional inquiry.

Recent advances have indicated that the interaction among multiple phytohormones, such as auxin, ethylene, and nitric oxide (NO), plays an important role in iron deficiency responses in plants (Schmidt et al., 2000; Seguela et al., 2008; Romera et al., 2011; Hindt and Guerinot, 2012). For example, recent reports indicate that auxin can regulate plant responses to iron deficiency through a NO-mediated signaling pathway (Chen et al., 2010; Jin et al., 2011). Furthermore, decreased auxin concentrations and polar auxin transport in auxin transporter mutants trigger up-regulation of iron deficient responsive genes (Xu et al., 2014). Our previous study demonstrates that AtGRXS17 is crucial for auxin response and function in temperature stress (Cheng et al., 2011). Whether AtGRXS17 mediates its effects on the iron deficiency response via modulation of auxin response pathways remains to be further investigated.

AtGRX7 is part of the ensemble of plant genes that sense and respond to fluctuations in iron availability. Using heterologous expression and reverse genetic approaches, this work establishes that AtGRXS17 functions under iron limiting conditions to modulate plant growth, iron accumulation, and redox balance.

NC and MG designed the study and wrote the paper. HY, JY, JD, YS, ST, TR, D-LW, JL, SP, and NC performed and analyzed the experiments. PN, EC, and KH provided technical assistance and analysis and interpretation of data. All authors reviewed the results and approved the final version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.