Increased Sucrose Accumulation Regulates Iron-Deficiency Responses by Promoting Auxin Signaling in Arabidopsis Plants

Abstract

Previous studies have identified that auxins acts upstream of nitric oxide in regulating iron deficiency responses in roots, but the upstream signaling molecule of auxins remains unknown. In this study, we showed that Fe deficiency increased sucrose (Suc) level in roots of Arabidopsis (Arabidopsis thaliana). Exogenous application of Suc further stimulated Fe deficiency-induced ferric-chelate-reductase (FCR) activity and expression of Fe acquisition-related genes FRO2, IRT1, and FIT in roots. The opposite patterns were observed in the dark treatment. In addition, FCR activity and expression of Fe acquisition-related genes were higher in the Suc high-accumulating transgenic plant 35S::SUC2 but were lower in the Suc low-accumulating mutant suc2-5 compared with wild-type plants under Fe-deficient conditions. Consequently, Fe deficiency tolerance was enhanced in 35S::SUC2 but was compromised in suc2-5. Exogenous Suc also increased root β-glucuronidase (GUS) activity in auxin-inducible reporter DR5-GUS transgenic plants under Fe deficiency. However, exogenous Suc failed to increase FCR activity and expression of Fe acquisition-related genes in the auxin transport-impaired mutants aux1-7 and pin1-1 as well as in the wild-type plants treated with an auxin transport inhibitor under Fe deficiency. In summary, we found that increased Suc accumulation is required for regulating Fe deficiency responses in plants, with auxins acting downstream in transmitting the Fe deficiency signal.

Figure 1.

Effect of Fe deficiency on sugar concentration in the roots of Col-0 in Arabidopsis. Five-week-old plants were grown in either complete (+ Fe) or Fe-free (-Fe) nutrient solutions. A, Concentrations of Suc, Fru (B), and Glc (C) in the roots were analyzed at 8, 12, 24, 48, and 96 h after each treatment. Data are expressed as mean ± sd (n = 4). An asterisk indicates significant differences between two treatments at each time point (one-way ANOVA, P < 0.05).

Figure 2.

Effects of carbon metabolites on the activity of root FCR in Col-0 Arabidopsis. The 5-week-old plants were grown in either complete (+ Fe) or Fe-free (- Fe) nutrient solutions. On day 3, the above nutrient solutions were supplied with or without various carbon metabolites, and the plants were continuously grown for 24 h. A, Relative root FCR activity of the plants in response to varying doses of exogenous Suc. B, Relative FCR activity in roots of the plants in response to various carbon metabolites. C, Visualization of FCR activity in roots with ferrozine in the agar medium. The concentrations of carbon metabolites used in experiments A and B are indicated in the figures. The concentration of Suc used in experiment C is 2 mM. Data are means ± sd (n = 5). Different letters indicate significant differences among treatments (one-way ANOVA, P < 0.05).

Figure 3.

Effects of dark treatment and exogenous Suc application on Fe deficiency responses in roots of Col-0 Arabidopsis. The 5-week-old plants were cultured in either complete (+ Fe) or Fe-free (- Fe) nutrient solutions. On day 3, a part of the plants was covered using a black box, and the above nutrient solutions were supplied with or without 2 mm Suc and the plants were continuously grown for 24 h. A, Relative FCR activity. B, Expression of FRO2. C, Expression of IRT1. Gene expression was analyzed by real-time qPCR. Transcript level of UBQ10 was used as an internal control. Data are means ± sd (n = 7). Different letters indicate significant differences among treatments (one-way ANOVA, P < 0.05).

Figure 4.

Comparison of Fe deficiency responses in roots between Col-0 plants and the suc2-5 mutants. The plants were precultured as described in “Materials and Methods.” Then the 20-d-old plants were transferred to either complete (+Fe) or Fe-free (- Fe) agar medium supplied with or without 2 mm Suc for 5 d. A, Relative FCR activity. B, Expression of FRO2. C, Expression of IRT1. Gene expression was analyzed by real-time qPCR. Transcript level of UBQ10 was used as an internal control. Data are means ± sd (n = 5-7). Different letters indicate significant differences among treatments with in a genotype (one-way ANOVA, P < 0.05). An asterisk shows a significant genotype by treatment interaction (two-way ANOVA, P < 0.05).

Figure 5.

Comparison of Fe deficiency responses in roots between Col-0 plants and the 35S::SUC2 transgenic plants. The indicated plants were treated as in Figure 1. A, Relative FCR activity. B, Expression of FRO2. C, Expression of IRT1. Gene expression was analyzed by real-time qPCR. Transcript level of UBQ10 was used as an internal control. Data are means ± sd (n = 5-7). Different letters indicate significant differences between two genotypes with in a treatment (one-way ANOVA, P < 0.05). An asterisk shows a significant genotype by treatment interaction (two-way ANOVA, P < 0.05).

Figure 6.

Fe deficiency tolerance of Col-0 plants and the suc2-5 mutants. The indicated plants were treated as in Figure 4. A, Photographs of shoots. B, Chlorophyll concentration of leaves. C, Fe level of plants. Data are means ± sd (n = 5). Different letters indicate significant differences between two genotypes with in a treatment (one-way ANOVA, P < 0.05). Asterisk and ns indicate that the genotype by treatment interactions are significant and not significant, respectively (two-way ANOVA, P < 0.05).

Figure 7.

Fe deficiency tolerance of Col-0 plants and the 35S::SUC2 transgenic plants. The 5-week-old plants were grown in nutrient solutions containing either 50 μm (+Fe) or 1 μm Fe-EDTA for 10 d. A, Photographs of leaves. B, Chlorophyll concentration of leaves. C, Fe level in roots and shoots. Data are means ± sd (n = 5). Different letters indicate significant differences between two genotypes with in a treatment (one-way ANOVA, P < 0.05). An asterisk shows a significant genotype by treatment interaction (two-way ANOVA, P < 0.05).

Figure 8.

The role of FIT in exogenous Suc regulation of Fe deficiency responses in roots. A, Comparison of FIT expression between Col-0 plant and the 35S::SUC2 transgenic plants. B, Effects dark treatment on FIT expression in Col-0 plant. C, Comparison of FIT expression between Col-0 plants and the suc2-5 mutants. D-F, Effects of exogenous Suc on FCR activity and the expression of FRO2 and IRT1 in Col-0 plants and the fit mutants. For the experiments in A-C, the plants were treated as in Figures 5, 3, and 4, respectively. For the experiments in D-F, the plants were treated as in Figure 2C. Gene expression was analyzed by real-time qPCR. Transcript level of UBQ10 was used as an internal control. Data are means ± sd (n = 5-7). Different letters indicate significant differences among treatments with in a genotype (one-way ANOVA, P < 0.05). An asterisk shows a significant genotype by treatment interaction (two-way ANOVA, P < 0.05).

Figure 9.

Effects of exogenous Suc on Fe deficiency responses in roots of Col-0 plant treated with auxin transport inhibitor. The 5-week-old plants were cultured in either complete (+ Fe) or Fe-free (- Fe) nutrient solution with or without addition of auxin transport inhibitor NPA (5 μM). On day 3, the above nutrient solutions were supplied with or without 2 mm Suc, and the plants were continuously grown for 24 h. A, Relative FCR activity. B-D, Expression of FRO2, IRT1, and FIT. Gene expression was analyzed by real-time qPCR. Transcript level of UBQ10 was used as an internal control. Data are means ± sd (n = 5-7). Different letters indicate significant differences among treatments (one-way ANOVA, P < 0.05).

Figure 10.

Effect of exogenous Suc on Fe deficiency responses in roots of Col-0 plant and the aux1-7 and pin1-1 mutants. The 5-week-old plants were grown in Fe-free (- Fe) nutrient solution. On day 3, the above nutrient solution was supplied with or without 2 mm Suc, and the plants were continuously grown for 24 h. A, Relative FCR activity. B-D, Expression of FRO2, IRT1, and FIT. Gene expression was analyzed by real-time qPCR. Transcript level of UBQ10 was used as an internal control. Data are means ± sd (n = 5-7). Different letters indicate significant differences between two treatments with in a genotype (one-way ANOVA, P < 0.05). An asterisk shows a significant genotype by treatment interaction (two-way ANOVA, P < 0.05).

Figure 11.

Schematic model of Suc in the regulation of Fe deficiency responses.