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. 2018 Sep 11:9:1325.
doi: 10.3389/fpls.2018.01325. eCollection 2018.

A Shoot Fe Signaling Pathway Requiring the OPT3 Transporter Controls GSNO Reductase and Ethylene in Arabidopsis thaliana Roots

María J García  1 , Francisco J Corpas  2 , Carlos Lucena  3 , Esteban Alcántara  3 , Rafael Pérez-Vicente  1 , Ángel M Zamarreño  4 , Eva Bacaicoa  4 , José M García-Mina  4 , Petra Bauer  5 , Francisco J Romera  3
Affiliations

A Shoot Fe Signaling Pathway Requiring the OPT3 Transporter Controls GSNO Reductase and Ethylene in Arabidopsis thaliana Roots

María J García et al. Front Plant Sci. .

Abstract

Ethylene, nitric oxide (NO) and glutathione (GSH) increase in Fe-deficient roots of Strategy I species where they participate in the up-regulation of Fe acquisition genes. However, S-nitrosoglutathione (GSNO), derived from NO and GSH, decreases in Fe-deficient roots. GSNO content is regulated by the GSNO-degrading enzyme S-nitrosoglutathione reductase (GSNOR). On the other hand, there are several results showing that the regulation of Fe acquisition genes does not solely depend on hormones and signaling molecules (such as ethylene or NO), which would act as activators, but also on the internal Fe content of plants, which would act as a repressor. Moreover, different results suggest that total Fe in roots is not the repressor of Fe acquisition genes, but rather the repressor is a Fe signal that moves from shoots to roots through the phloem [hereafter named LOng Distance Iron Signal (LODIS)]. To look further in the possible interactions between LODIS, ethylene and GSNOR, we compared Arabidopsis WT Columbia and LODIS-deficient mutant opt3-2 plants subjected to different Fe treatments that alter LODIS content. The opt3-2 mutant is impaired in the loading of shoot Fe into the phloem and presents constitutive expression of Fe acquisition genes. In roots of both Columbia and opt3-2 plants we determined 1-aminocyclopropane-1-carboxylic acid (ACC, ethylene precursor), expression of ethylene synthesis and signaling genes, and GSNOR expression and activity. The results obtained showed that both 'ethylene' (ACC and the expression of ethylene synthesis and signaling genes) and 'GSNOR' (expression and activity) increased in Fe-deficient WT Columbia roots. Additionally, Fe-sufficient opt3-2 roots had higher 'ethylene' and 'GSNOR' than Fe-sufficient WT Columbia roots. The increase of both 'ethylene' and 'GSNOR' was not related to the total root Fe content but to the absence of a Fe shoot signal (LODIS), and was associated with the up-regulation of Fe acquisition genes. The possible relationship between GSNOR(GSNO) and ethylene is discussed.

Keywords: S-nitrosoglutathione (GSNO); S-nitrosoglutathione reductase (GSNOR); ethylene; glutathione (GSH); iron; long distance iron signal (LODIS); nitric oxide (NO); phloem.

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Figures

FIGURE 1
FIGURE 1
Effect of Fe deficiency and foliar application of Fe on the expression of (A) Fe acquisition genes FRO2, IRT1, and FIT; and (B) ethylene synthesis genes SAM1, ACS6 and ACO2, in roots of Arabidopsis WT Columbia plants. Plants were grown in nutrient solution with Fe (+Fe). Some of them were transferred to nutrient solution without Fe 3 days before harvest (–Fe). Half of the –Fe plants were sprayed with Fe 24 h before harvest (–Fe+fol Fe). Relative expression was calculated in relation to +Fe. Data represent the mean ± SE of three independent biological replicates. Within each gene, bars with different letters indicate significant differences (P < 0.05).
FIGURE 2
FIGURE 2
Effect of Fe deficiency and foliar application of Fe on ACC content in roots of Arabidopsis WT Columbia and opt3-2 mutant plants. Treatments as in Figure 1. Data represent the mean ± SE of six replicates. Within each genotype, bars with different letters indicate significant differences (P < 0.05). Significant difference between the +Fe treatment from opt3-2 and Col is also indicated: ∗∗P < 0.05.
FIGURE 3
FIGURE 3
ACC concentration in Fe-sufficient roots (A) and shoot appearance (B) of Arabidopsis WT Columbia and opt3-2, frd3-3, and nas4x-1 mutant plants. Note the chlorosis in the frd3-3 and nas4x-1 mutants. Plants were grown in nutrient solution with Fe. Data represent the mean ± SE of six replicates. Significant differences from Col are indicated: ∗∗P < 0.05.
FIGURE 4
FIGURE 4
Expression of ethylene synthesis genes (A) SAM1, (B) ACS6 and (C) ACO2, and ethylene signaling genes (D) EIN2, (E) EIN3 and (F) EIL1, in Fe-sufficient roots of Arabidopsis WT Columbia and opt3-2 mutant plants. Plants were grown in nutrient solution with Fe. Relative expression was calculated in relation to Col. Data represent the mean ± SE of three independent biological replicates. Significant differences between opt3-2 and Col are indicated: ∗∗P < 0.05.
FIGURE 5
FIGURE 5
Time course experiment of the effect of Fe deficiency on the expression of (A) GSNOR1, Fe acquisition genes (B) FIT, (C) bHLH38, (D) IRT1 and (E) FRO2), and on (F) ferric reductase activity, in roots of Arabidopsis WT Columbia plants. Plants were grown in nutrient solution with Fe (+Fe). Some of them were transferred to nutrient solution without Fe (–Fe) 6, 12, 24, 48, or 72 h before determination of ferric reductase activity or harvest. For genes, data represent the mean ± SE of three independent biological replicates. Relative expression was calculated in relation to +Fe. For ferric reductase activity, data represent the mean ± SE of six replicates.
FIGURE 6
FIGURE 6
Effect of Fe deficiency and foliar application of Fe on GSNOR activity in roots of Arabidopsis WT Columbia plants. Plants were grown in nutrient solution with Fe (+Fe) and some of them were transferred to nutrient solution without Fe 2 days before harvest (–Fe). Half of the –Fe plants were sprayed with Fe 24 h before harvest (–Fe+fol Fe). Data represent the mean ± SE of six replicates. Bars with different letters indicate significant differences (P < 0.05).
FIGURE 7
FIGURE 7
GSNOR1 expression in roots of Fe-sufficient Arabidopsis WT Columbia and opt3-2 mutant plants. Plants were grown in nutrient solution with Fe. Relative expression was calculated in relation to Col. Data represent the mean ± SE of three independent biological replicates. Significant difference between opt3-2 and Col is indicated: ∗∗P < 0.05.
FIGURE 8
FIGURE 8
Effect of P or S deficiency on GSNOR1 expression in roots of Arabidopsis WT Columbia plants. Plants were grown in complete nutrient solution (Control). Some of them were transferred to nutrient solution without P (–P) or without S (–S) during the last 2 days. Relative expression was calculated in relation to Control. Data represent the mean ± SE of three independent biological replicates. Significant differences from the Control treatment are indicated: ∗∗P < 0.05.
FIGURE 9
FIGURE 9
Effect of ACC on GSNOR1 expression in roots of Arabidopsis WT Columbia plants. Plants were grown in nutrient solution with 40 μM Fe (+Fe) and half of them treated with 1 μ M ACC, final concentration (+Fe+ACC), 24 h before harvest. Relative expression was calculated in relation to +Fe. Data represent the mean ± SE of three independent biological replicates. Significant differences from the +Fe treatment are indicated: ∗∗P < 0.05.
FIGURE 10
FIGURE 10
Effect of Fe deficiency and foliar application of Fe on GSNO content in roots of Arabidopsis WT Columbia and opt3-2 mutant plants. Plants were grown in nutrient solution with Fe (+Fe). Some of them were transferred to nutrient solution without Fe 2 days before harvest (–Fe). Half of the –Fe plants were sprayed with Fe 24 h before harvest (–Fe+fol Fe). Data represent the mean ± SE of six replicates. Within each genotype, bars with different letters indicate significant differences (P < 0.05). Significant difference between the +Fe treatment from opt3-2 and Col is also indicated: ∗∗P < 0.05.
FIGURE 11
FIGURE 11
Effect of Fe deficiency on GSNO content in roots of pea WT Sparkle and dgl mutant plants. Plants were grown in nutrient solution with Fe (+Fe) and half of them were transferred to nutrient solution without Fe 2 days before harvest (–Fe). Data represent the mean ± SE of six replicates. Within each genotype, bars with different letters indicate significant differences (P < 0.05). Significant difference between the +Fe treatment from dgl and Sparkle is also indicated: ∗∗P < 0.05.
FIGURE 12
FIGURE 12
Working Model to explain the role of LODIS on the regulation of Fe acquisition genes. Once inside roots, Fe is translocated to leaves through the xylem, bound to citrate (provided by the FRD3 transporter). In shoots, some Fe (either as free ions or in chelated form) can enter the phloem through the OPT3 transporter, and moves back to roots bound to a chelating agent (forming LODIS). In roots, LODIS could negatively affect ethylene synthesis and signaling, and GSNOR expression and/or activity, which can lead to enhanced GSNO. Besides LODIS, which would act as a repressor of Fe responses, some other shoot signals, like sucrose and auxin, would act as activators of Fe responses through NO (Lin et al., 2016). The possible relationship of ethylene, NO, GSH, GSNOR, and GSNO is depicted in more detail in Figure 13. In green are components whose expression, activity and/or content is known to increase under Fe deficiency while in red are components whose expression, activity, and/or content is known to increase under Fe sufficiency. chel, chelating agent; GSH, glutathione; GSNO, S-nitrosogluthatione; GSNOR, GSNOR reductase; ET, ethylene; ETresp, ethylene response; Met, methionine (→: promotion; T¯: inhibition).
FIGURE 13
FIGURE 13
Model to explain the relationship between ethylene and NO, and between ethylene and GSNOR (GSNO), in roots. In previous works, and also in this work (see Figure 2 and Table 2), it has been shown that ethylene, NO, and GSH increase in Fe-deficient roots of dicot plants (see section “Introduction”). Moreover, it has been shown that ethylene can influence NO accumulation in roots and vice versa (black arrows; García et al., 2011; Liu et al., 2017). In this work, it has been found that ethylene and GSNOR increase under low LODIS accumulation in roots (see Figure 12). After that, a new model, complementary of the previous one, is proposed (blue arrows) suggesting mutual possible interactions among ethylene, GSNOR, GSH and NO: (1) ethylene can upregulate GSNOR (see Figure 9); (2) GSNOR can decrease GSNO (Corpas et al., 2013); (3) Low GSNO can unblock ethylene synthesis since nitrosylation of SAM synthetase by GSNO can inhibit ethylene synthesis (Lindermayr et al., 2006; Freschi, 2013; see section “Discussion”); (4) GSNO derives from NO and GSH (Corpas et al., 2013); (5) GSH can enhance ethylene production (Datta et al., 2015). In green are components whose expression, activity and/or content is known to increase under Fe deficiency while in red are components whose content is known to decrease under Fe deficiency. GSH, glutathione; GSNO, S-nitrosoglutathione; GSNOR, GSNO reductase; ET, ethylene; NO, nitric oxide (→: promotion; T¯: inhibition).
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