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. 2017 Jan 8;119(6):945–956. doi: 10.1093/aob/mcw250

Gibberellins regulate iron deficiency-response by influencing iron transport and translocation in rice seedlings (Oryza sativa)

Baolan Wang 1,2, Haifang Wei 1,3, Zhen Xue 4, Wen-Hao Zhang 1,2,3,*
PMCID: PMC5604592  PMID: 28065924

Abstract

Background and aims Gibberellins (GAs) are a class of plant hormones with diverse functions. However, there has been little information on the role of GAs in response to plant nutrient deficiency.

Methods To evaluate the roles of GAs in regulation of Fe homeostasis, the effects of GA on Fe accumulation and Fe translocation in rice seedlings were investigated using wild-type, a rice mutant (eui1) displaying enhnaced endogenous GA concentrations due to a defect in GA deactivation, and transgenic rice plants overexpressing OsEUI.

Key Results Exposure to Fe-deficient medium significantly reduced biomass of rice plants. Both exogenous application of GA and an endogenous increase of bioactive GA enhanced Fe-deficiency response by exaggerating foliar chlorosis and reducing growth. Iron deficiency significantly suppressed production of GA1 and GA4, the biologically active GAs in rice. Exogenous application of GA significantly decreased leaf Fe concentration regardless of Fe supply. Iron concentration in shoot of eui1 mutants was lower than that of WT plants under both Fe-sufficient and Fe-deficient conditions. Paclobutrazol, an inhibitor of GA biosynthesis, alleviated Fe-deficiency responses, and overexpression of EUI significantly increased Fe concentration in shoots and roots. Furthermore, both exogenous application of GA and endogenous increase in GA resulting from EUI mutation inhibited Fe translocation within shoots by suppressing OsYSL2 expression, which is involved in Fe transport and translocation.

Conclusions The novel findings provide compelling evidence to support the involvement of GA in mediation of Fe homeostasis in strategy II rice plants by negatively regulating Fe transport and translocation.

Keywords: Rice (Oryza sativa), Fe deficiency, strategy II plant, gibberellins, Fe transport and translocation

INTRODUCTION

Iron (Fe) is an essential nutrient required for plant growth and development. Iron availability is often low in soils due to its low solubility, especially in calcareous soils, leading to limited plant growth (Romera and Alcántara, 2004). Plants have evolved two contrasting strategies to cope with Fe deficiency in soils. Strategy I occurs in non-graminaceous monocots and dicots, while strategy II exists in graminaceous monocots (Römheld and Marschner, 1986; Kobayashi and Nishizawa, 2012). Strategy I plants are distinguished by enhanced ferric reductase activity, acidification of the rhizosphere and up-regulation of Fe2+ transporters upon exposure to Fe-deficient medium (Curie and Briat, 2003; Hell and Stephan, 2003; Romera and Alcántara, 2004). In contrast, Strategy II plants often show enhanced secretion of phytosiderophores (PSs), which belong to the mugineic acid (MA) family, to form a Fe3+-MA complex in response to Fe deficiency (Kobayashi and Nishizawa, 2012). MAs are synthesized from three molecules of S-adenosyl-methionine by three sequential enzymes: nicotianamine (NA) synthase (NAS), NA aminotransferase (NAAT) and deoxymugineic acid synthase (DMAS). In rice, genes encoding these enzymes have been identified (Inoue et al., 2003). The Fe3+-MA is transported into root cells by a membrane transporter of Yellow Stripe1 (YS1) in maize (von Wiren et al., 1994; Curie et al., 2001). For rice, the secretion of MAs from roots to the rhizosphere is mediated by OsTOM1 (Nozoye et al., 2011), and the resulting Fe3+-MA complexes are taken up into root cells by OsYSL15, a yellow-stripe like (YSL) transporter in the plasma membrane (Lee et al., 2009). In addition to the YSL system, rice plants can also acquire Fe2+ through IRT transporters, and two genes encoding the Fe2+ transporters, OsIRT1 and OsIRT2, have been isolated (Bughio et al., 2002; Ishimaru et al., 2006). OsYSL2 is a rice metal-NA transporter responsible for translocation of Fe and Mn in shoots via the phloem, and it also plays roles in the mediation of transport of Fe from roots to shoots (Koike et al., 2004; Ishimaru et al., 2010). In addition, several transcription factors that are involved in regulation of Fe homeostasis have been isolated in rice. These include OsIRO2, OsIRO3, OsIDEF1, OsIDEF2 and OsbHLH133. For instance, OsIRO2 is synchronously expressed with genes involved in Fe homeostasis, and responsible for rice growth and yield in calcareous soils (Ogo et al., 2007). Despite identification of a number of key genes involved in Fe homeostasis in rice, the overall signalling network associated with Fe-deficiency responses remains largely to be dissected.

Numerous studies have shown that plant hormones play important roles in responses to stresses associated with deficiency and/or toxicity of mineral nutrients. For instance, auxin, ethylene, cytokinins and nitric oxide (NO) have been reported to be involved in the regulation of Fe deficiency-induced physiological processes in strategy I plants (Ivanov et al., 2012). Although there are reports indicating that ethylene is not involved in the regulation of Fe-deficiency responses by strategy II plants (Romera et al., 1999; Romera and Alcántara, 2004), Wu et al. (2011) showed that ethylene regulates the expression of genes associated with Fe acquisition in rice plants. As rice plants have both strategy I and II systems for Fe acquisition, it is likely that, similar to strategy I plants, plant hormones may also play a regulatory role in mediation of Fe acquisition by rice plants. However, in contrast to strategy I plants, there has been limited information on the roles of phytohormones in response of strategy II plants to Fe deficiency. We recently demonstrated that brassinosteroids are involved in Fe homeostasis in rice (Wang et al., 2015).

Gibberellins (GAs) are one of the classical plant hormones with multiple functions in the regulation of physiological processes associated with developmental and environmental cues. For example, Jiang et al. (2007) reported that GA is involved in the regulation of response to phosphate deficiency in Arabidopsis. Matsuoka et al. (2014) reported that expression of many genes associated with Fe-deficiency responses was up-regulated in a GA-deficient Arabidopsis mutant, a strategy I plant, by exogenous application of GA. Very recently, Wild et al. (2016) showed that the spatial distribution of GA-regulated DELLA growth repressors in Arabidopsis roots was adapted to the root system architecture and the Fe-uptake machinery to the plant’s Fe demand. Given the differences in Fe mobilization and transport between strategy I and strategy II plants, whether and how GA plays a role in Fe-deficiency responses in strategy II plants remain to be investigated.

Studies on GA function have been greatly promoted by isolation of numerous rice mutants associated with GA metabolism and signalling (Tong et al., 2014). Among the mutants, the recessive tall rice mutant elongated uppermost internode (eui) exhibits a rapid and enhanced elongation of the internode and increased endogenous GA concentrations. The EUI gene encodes a GA-deactivating enzyme, and overexpression of EUI results in a marked inhibition of plant growth (Luo et al., 2006; Zhu et al., 2006). In the present study, we used wild-type (WT), eui1 mutant and EUI overexpression line to evaluate the role of GA in the response of strategy II rice plants to Fe deficiency. We also monitored the changes in endogenous active GA levels in WT rice plants in response to Fe deficiency. Our results show that Fe deficiency significantly inhibited GA1 and GA4 production in WT rice plants, and that GA negatively modulates Fe utilization in rice seedlings by reducing Fe transport and translocation in shoots.

MATERIALS AND METHODS

Plant growth and treatments

Rice seeds of Zhonghua 10, ZS97, eui1 and EUI-overexpression (EUI-OE) lines were surface-sterilized by incubation for 3 min in 75 % ethanol, followed by 10 min in 0·1 % HgCl2, and then washed thoroughly with sterile water. Seeds of the eui1 mutant and EUI-OE were kindly provided by Professor Z. H. He at Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. GC-MS analysis showed that eui1 plants contained a much higher level of GA1 and GA4 than did wild-type plants, and the EUI-OE plants had an extremely dwarf phenotype (Zhu et al., 2006). The sterilized seeds were soaked in water for 24 h in the dark and then transferred to a nylon net floating on water for 1 week. Then, the 7-d-old seedlings were transferred to nutrient solution containing one-half-strength Kimura B solution. The nutrient solution contained the macronutrients (mm) (NH4)2SO4 (0·18), MgSO4·7H2O (0·27), KNO3 (0·09), K2SO4 (0·47), Ca(NO3)2·4H2O (0·18) and KH2PO4 (0·09), and the micronutrients (µm) MnCl2·4H2O (0·9), H3BO3 (46·2), H2MoO4 (0·36), ZnSO4·7H2O (0·77) and CuSO4·5H2O (0·32), supplied with 50 µm Fe (III)-EDTA with pH of 5·5, and the nutrient solution pH was adjusted daily and was renewed every 3 d. The hydroponic experiments were carried out in a growth room with a 16-h light (30 °C)/8-h dark (22 °C) photoperiod, and the relative humidity was controlled at approx. 70 %. After 1 week preculture, the rice plants were exposed to Fe-sufficient [100 µm Fe (III)-EDTA[ or Fe-deficient [0 µm Fe (III)-EDTA] full-strength Kimura B solution with and without 0·1 µm gibberellic acid (GA3) or 1 µm paclobutrazol (PAC) for 1 week.

Measurement of chlorophyll concentration

After treatments with different levels of Fe and GA/PAC supply for 7 d, the fully expanded youngest leaves were used to determined total chlorophyll concentration (SPAD values) with a portable SPAD meter (SPAD-502) (Zhejiang Top Instrument Co., Ltd, China).

Determination of plant growth and analysis of metal concentration

After treatments with different levels of Fe and GA/PAC supply for 7 d, shoot length was recorded by a ruler, and seedlings were harvested and separated into leaves, stems and roots. Fresh biomass of different tissues was measured and the tissues were then dried for 2 d at 75 °C for determination of dry biomass. After measurements of biomass, leaf, stem and root samples were digested with concentrated nitric acid and hydrogen peroxide. The total Fe concentrations were determined by inductively coupled plasma emission spectrometry (ICP-OES).

Determination of endogenous bioactive GA concentrations

The endogenous concentrations of bioactive GA1 and GA4 in leaves and stems of WT rice plants were determined by GC-MS following a modification of the protocols described by Wijayanti et al. (1995). Briefly, rice leaves and stems (300 mg) were collected, instantly frozen in liquid nitrogen and ground with a pestle. To better preserve the GAs, the ground leaves and stems were dried under vacuum (0·08 mbar) at low temperature (−50 °C). The dried rice leaf and stem samples were weighed, recorded, extracted with MeOH/H2O = 4:1 containing [2H2] GA1 and [2H2] GA4 as internal standards and evaporated to dryness. The dried extract was re-dissolved in MeOH/H2O = 4:1 and passed through a C18 Sep-pack column and HPLC column of a ZORBAX SB-C18 device (150 mm × 4·6 mm, 5 μm). The fractions corresponding to GA1 and GA4 were collected and dried in a centrifugal concentrator. The GA fractions were trimethylsilylated with N-methyl-N-trimethylsilylteifluoroacetamide (MSTFA) at 80 °C for 30 min. The samples were taken to dryness and dissolved in hexane prior to injecting into a fused silica glass capillary column (DB-5, 0·25 mm × 30 m, 0·25 μm film thickness, J&W). Injection and interface temperatures were 260 and 280 °C, respectively. The column temperature was maintained at 80 °C for 2 min, and then increased by 10 °C min−1 to 180 °C, followed by 6 °C min−1 to 290 °C. Electron energy was 70 eV. The identity of GAs was verified by monitoring diagnostic ions of both endogenous and deuterated GAs according to the reference of Fernández et al. (1997). The internal standards [2H]2-GA1 (cat. no. 032 2491) and [2H]2-GA4 (cat. no. 032 2531) were purchased from OlChemIm Company.

Expression of genes encoding Fe uptake and translocation

Total RNA was extracted from roots and shoots of rice plants subjected to Fe or GA treatments. Total RNA was isolated using RNAiso reagent (Takara) and was reverse-transcribed into first-strand cDNA with a PrimeScript RT reagent Kit with gDNA Erager (Takara). Real-time PCR was performed in an optical 96-well plate with an Applied Biosystems Stepone real-time PCR system. Each reaction contained 7·5 μL of 2× SYBR Green Master Mix reagent, 0·5 μL of cDNA samples and 0·6 μL of 10 µm gene-specific primers in a final volume of 15 μL. The thermal cycle used was as follows: 95 °C for 10 min; 40 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s. All the primers used for quantitative RT-PCR are listed in Supplementary Data Table S1. The relative quantification method (ΔΔCt) was used to evaluate quantitative variation between the replicates examined. The PCR signals were normalized to those of actin or rice polyubiquitin (RubQ1).

Statistical analysis

A two-way ANOVA was conducted using the SAS statistical software. Significant differences between treatments were evaluated by least significant difference multiple range tests (P ≤ 0·05).

RESULTS

Effects of exogenous application of GA and GA biosynthesis inhibitor on plants

To test whether GA is involved in Fe-deficiency-induced changes in physiological processes in rice plants, the effects of exogenous application of GA on growth and chlorophyll (CHL) concentration of rice seedlings grown in Fe-sufficient and Fe-deficient media were studied. An apparent chlorosis was observed in the newly formed leaves of rice seedlings grown in Fe-deficient medium for 1 week (Fig. 1A). Accordingly, exposure of rice plants to Fe deficiency led to a significant decrease in CHL concentration (Fig. 1B). Furthermore, the chlorosis became more evident by application of GA to Fe-deficient rice seedlings (Fig. 1A). In contrast, application of GA had no apparent effect on leaf chlorosis in rice seedlings grown in Fe-sufficient medium (Fig. 1A). In addition to exogenous GA application, we further evaluated the effect of PAC, an inhibitor of GA biosynthesis, on plant growth and CHL concentration. Iron-deficiency-induced chlorosis became less visible when PAC was added to Fe-deficient medium (Fig. 1A). Accordingly, we found that treatment with PAC led to a significant increase in CHL concentration of Fe-deficient seedlings, and that the same PAC treatment also enhanced CHL concentration in Fe-sufficient rice seedlings (Fig. 1B). There was a significant interaction between Fe treatments and GA-related treatments in CHL concentration (P = 0·0011).

Fig. 1.

Fig. 1.

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Effect of iron, GA and PAC treatments on leaf chlorophyll concentrations. One-week-old rice plants (Zhonghua 10) pre-cultured with Fe (III)-EDTA were exposed to Fe-sufficient [100 µm Fe (III)-EDTA] or Fe-deficient [0 µm Fe (III)-EDTA] conditions with or without 0·1 µm GA or 1 µm PAC for 1 week. Chlorophyll concentrations of the fully expanded youngest leaf were measured using a SPAD chlorophyll meter. Data are means±s.d. (n = 10). Different capital letters indicate a significant difference (P ≤ 0·05) between +Fe and –Fe treatment for the tree regimes (control, + GA and +PAC), and different lowercase letters denote a significant difference (P ≤ 0·05) among the three treatments under the same Fe conditions.

In addition to CHL concentration, Fe deficiency also inhibited shoot elongation and plant growth (Fig. 2). Application of GA significantly increased shoot length under both Fe-sufficient and Fe-deficient conditions (Fig. 2A). Since GA reduced CHL concentration by about 40 % (Fig. 1), but increased shoot length (Fig. 2C), we further assessed the effects of GA on leaf, stem and root growth under Fe-sufficient and Fe-deficient conditions. Similar to CHL concentration, exposure to Fe-deficient medium led to a significant reduction in leaf, stem and root biomass. In contrast, GA application had no effect on biomass of leaf, stem and root of Fe-sufficient rice seedlings (Fig. 2B–D). In contrast to leaf biomass, biomass of stem and root was not affected by GA under Fe-deficient conditions (Fig. 2C, D). Application of PAC significantly suppressed shoot length and reduced plant biomass, irrespective of Fe supply (Fig. 2). For example, biomass of leaf and stem was significantly reduced by PAC treatment under both Fe-sufficient and Fe-deficient conditions (Fig. 2B, C). However, the magnitude of the inhibition in Fe-deficient seedlings was less than that in Fe-sufficient seedlings.

Fig. 2.

Fig. 2.

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Effect of Fe, GA and PAC treatments on plant growth. One-week-old rice plants (Zhonghua 10) pre-cultured with Fe (III)-EDTA were exposed to Fe-sufficient [100 µm Fe (III)-EDTA] or Fe-deficient [0 µm Fe (III)-EDTA] conditions with or without 0·1 µm GA or 1 μm PAC for 1 week. Shoot (A) length and biomass of different organs (B, C and D) were measured. Data are means±s.d (n = 9 for shoot length, and n = 5 for biomass). Treatments and statistical analysis are as detailed in Fig. 1.

Endogenous bioactive GA concentrations in leaf and stem of WT plants were reduced by Fe deficiency

The observations that exogenous application of GA enhanced Fe-deficiency-induced leaf chlorosis of rice plants imply that GA levels may play an important role in Fe status in planta. To test if Fe status also affects bioactive GA production, we monitored the effect of Fe deficiency on concentrations of the two bioactive GAs (GA1 and GA4) in leaves and stems of WT plants. As shown in Fig. 3, exposure of WT rice plants to Fe-deficient medium led to significant reductions in GA1 and GA4 concentrations in leaves and stems (Fig. 3). This result confirms that Fe status is involved in GA biosynthesis in rice plants.

Fig. 3.

Fig. 3.

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Effect of Fe deficiency on endogenous bioactive GA production in leaf (A) and stem (B). One-week-old rice plants of WT (ZS 97) pre-cultured with Fe (III)-EDTA were exposed to Fe-sufficient [100 µm Fe (III)-EDTA] or Fe-deficient [0 µm Fe (III)-EDTA] conditions for 1 week. Bioactive GA in leaf and stem was measured. Data are means±s.d (n = 4 or 5). Means with different letters denote significant difference between +Fe and –Fe treatments (P ≤ 0·05).

The eui1 mutant and EUI-OE line differed in their responses to Fe deficiency

EUI1 encodes a cytochrome P450 monooxygenase that epoxidizes GA in a novel deactivation reaction in rice, and mutation of EUI confers a higher active GA concentration in planta in the eui1 mutant (Luo et al., 2006; Zhu et al., 2006). To further investigate the interaction among GA and Fe, rice mutant eui1 and transgenic rice line EUI-OE were used. No significant differences in CHL concentration among the mutant, EUI-OE line and WT plants were detected when grown in Fe-sufficient medium (Fig. 4A). Exposure of these plants to Fe-deficient medium led to a significant reduction in foliar CHL in WT, eui1 and EUI-OE plants, with the magnitude of reduction following the order eui1 > WT > EUI-OE (Fig. 4A), indicating that rice plants with higher endogenous GA concentrations are more sensitive to Fe deficiency. These results are also in agreement with those of exogenous application of GA and PAC. EUI mutation significantly increased shoot length under both Fe-sufficient and Fe-deficient conditions, and overexpression of this gene inhibited shoot growth (Fig. 4B). Compared to the WT plant, mutation and overexpression of this gene significantly increased and decreased respectively the biomass of leaf and stem regardless of Fe supply (Table 1). Mutation of eui1 had no impact on root biomass, while EUI-OE reduced root biomass by 50 % (Table 1). Biomass of leaf and stem in WT, eui1 and EUI-OE plants was significantly reduced by exposure to Fe-deficient medium, while root biomass of WT, eui1 mutant and EUI-OE plants was similar (Table 1).

Fig. 4.

Fig. 4.

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Leaf chlorophyll and shoot length of WT (ZS 97), eui1 and EUI-OE rice plants grown in Fe-sufficient and Fe-deficient conditions. One-week-old rice plants of WT, eui1 and EUI-OE pre-cultured with Fe (III)-EDTA were exposed to Fe-sufficient [100 µm Fe (III)-EDTA] or Fe-deficient (0 µm Fe (III)-EDTA] conditions for 1 week. Leaf chlorophyll (A) and shoot length (B) were measured. Data are means±s.d (n = 10). Different capital letters denote significant difference (P ≤ 0·05) for the three plant materials (WT, eui1 and EUI-OE) between +Fe and –Fe treatments. Different lowercase letters denote significant difference (P ≤ 0·05) for the three plant materials within the same Fe treatments.

Table 1.

Biomass of different plant organs of WT (ZS 97), eui1 and EUI-OE rice plants grown in Fe-sufficient and Fe-deficient conditions

Plant organ Treatment Plant organs f. wt biomass (mg per plant)
WT eui EUI-OE
Leaf +Fe 76·35±1·58 *b 90·00±4·49 *a 24·40±2·64 c
–Fe 64·73±4·47 a 72·78±3·33a 20·10±1·77 b
Stem +Fe 80·77±2·26*b 108·87±4·30*a 29·97±4·56 c
–Fe 62·52±4·84 b 79·5±3·30 a 22·75±1·92 c
Root +Fe 35·00±1·04 a 33·42±2·67 a 17·83±2·46 b
–Fe 33·75±2·78 a 33·23±1·73 a 14·83±0·17 b
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Data are means±s.e.

*

Significant difference between +Fe and –Fe treatment within the same cultivar at the level of P ≤ 0·05. Means with different letters are significantly different (P ≤ 0·05) within three cultivars at the same Fe treatments.

Fe acquisition was reduced by both exogenous GA application and mutation of the EUI gene

In addition to CHL and plant growth, we evaluated the effects of GA on Fe concentrations in leaves, stems and roots. Exposure of rice seedlings to Fe-deficient medium led to a marked reduction of Fe concentrations in leaves, stems and roots of WT, eui1 and EUI-OE plants (Fig. 5). Exogenous application of GA reduced Fe concentration in leaf, while it had no effect on stem Fe concentration (Fig. 5). By contrast, exogenous application of GA markedly increased Fe concentration in roots under both Fe-sufficient and Fe-deficient conditions (Fig. 5). These results may imply that GA is involved in regulation of Fe translocation from roots to leaves. Compared to WT plants, mutation of EUI led to a significant reduction in Fe concentrations in leaves and stems, while Fe concentration in roots was not affected by the mutation, irrespective of Fe supply (Fig. 5). Overexpression of EUI in rice plants significantly increased Fe concentrations in leaves, stems and roots under both Fe-sufficient and Fe-deficient conditions (Fig. 5). There was a significant interaction between Fe treatments and GA-related treatments in root Fe concentration (P = 0·0074).

Fig. 5.

Fig. 5.

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Iron concentrations of different plant organs of WT (ZS 97), eui1 and EUI-OE rice plants grown in Fe-sufficient and Fe-deficient conditions. One-week-old rice plants of WT, eui1 and EUI-OE pre-cultured with Fe (III)-EDTA were exposed to Fe-sufficient [100 µm Fe (III)-EDTA] or Fe-deficient [0 µm Fe (III)-EDTA] for one week. Iron concentrations in different organs were measured. Data are means±s.d (n = 4). Different capital letters denote significant difference (P ≤ 0·05) for the three plant materials between +Fe and –Fe treatment. Different lowercase letters denote significant difference (P ≤ 0·05) for the three plant materials under the same levels of Fe supply.

Exogenous GA application reduced foliar CHL concentrations in eui1 mutant and EUI-OE plants

Exogenous application of GA enhanced Fe-deficiency-induced foliar chlorosis in WT plants. We further examined the responses of the eui1 mutant and EUI-OE plants to exogenous GA application under both Fe-sufficient and Fe-deficient conditions. Similar to WT plants, GA application had no impacts on foliar CHL concentrations in the eui1 mutant and EUI-OE plants under Fe-sufficient conditions (Fig. 6A, B), while exogenous application of GA significantly reduced foliar CHL concentrations in the eui1 and EUI-OE plants under Fe-deficient conditions (Fig. 6C, D). Furthermore, the magnitude of reduction in foliar CHL concentration was higher in the eui1 mutant than in WT plants. These results suggest that foliar CHL concentration may be negatively correlated with endogenous GA concentrations in leaves under Fe-deficient conditions.

Fig. 6.

Fig. 6.

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Effect of GA application on leaf chlorophyll of WT (ZS 97), eui1 and EUI-OE plants grown under Fe-sufficient and Fe-deficient conditions. One-week-old rice plants of WT, eui1 and EUI-OE pre-cultured with Fe (III)-EDTA were exposed to Fe-sufficient [100 µm Fe (III)-EDTA] or Fe-deficient [0 µm Fe (III)-EDTA] with or without 0·1 µm GA for 1 week. Leaf chlorophyll was measured. Data are means±s.d (n = 10). Treatments and statistical analysis are as detailed in Fig. 5.

Expression patterns of Fe deficiency-responsive genes were regulated by GA

Given the important role of transcription factor OsIRO2 in Fe homeostasis in rice (Ogo et al., 2007), we compared the responses of this gene in roots of WT, eui1 mutant and EUI-OE plants to exogenous application of GA under Fe-sufficient and Fe-deficient conditions at the transcriptional level. Neither exogenous application of GA nor mutation of the EUI gene had any impact on the expression level of OsIRO2 under Fe-sufficient conditions, but overexpression of EUI significantly depressed the expression of OsIRO2 under Fe-sufficient conditions (Fig. 7A). Iron deficiency up-regulated the expression of OsIRO2 in WT, eui1 mutant and EUI-OE plants (Fig. 7A). Moreover, exogenous application of GA and Fe deficiency had an additive effect on the expression of OsIRO2 (Fig. 7A). Compared to WT, the EUI mutation inhibited the expression of OsIRO2 under Fe-deficient conditions, and EUI-OE did not affect the expression of OsIRO2 under Fe-deficient conditions (Fig. 7A). In addition to OsIRO2, we also evaluated the effect of GA on the expression of genes related to MA biosynthesis and Fe3+-MA uptake in roots of WT, eui1 mutant and EUI-OE plants, including OsNAS1, OsNAS2, OsNAAT1, OsDMAS1 and OsYSL15. Similar to OsIRO2, exposure to Fe-deficient medium led to up-regulation of these genes (Fig. 7B–F). The expression levels of OsNAS1, OsNAS2 and OsDMAS1 in WT plants were not altered by addition of GA to the Fe-sufficient and Fe-deficient media, and only OsNAAT1 was up-regulated by GA under Fe-deficient conditions (Fig. 7B–E). In contrast, the expression of OsYSL15 in WT plants was enhanced by GA application regardless of Fe supply (Fig. 7F). Mutation of EUI enhanced expression of OsNAS1, OsNAS2 and OsNAAT1, while the transcript levels of OsDMAS1 and OsYSL15 remained unchanged by the mutation under Fe-sufficient conditions (Fig. 7B–F). The expression of OsNAS1 was suppressed by the EUI gene mutation under Fe-deficient conditions. Over-expression of EUI led to depressed expression of all these genes under Fe-sufficient conditions (Fig. 7B–F). Treatment of EUI-OE plants with Fe deficiency up-regulated expression of OsNAS1 and OsNAS2, while it suppressed expression of OsYSL15 with the expression of remaining genes unchanged (Fig. 7B–F). There was an extremely significant interaction between Fe treatments and GA-related treatments in the expression of OsIRO2, OsNAS1, OSNAS2 and OsYSL15 of root (P < 0·0001).

Fig. 7.

Fig. 7.

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Quantitative RT-PCR analysis of Fe uptake-related genes in roots. Data are means±s.e. of three biological replicates. Different capital letters denote significant difference (P ≤ 0·05) for WT, WT/GA, eui1 and EUI-OE between +Fe and –Fe treatments. Different lowercase letters denote significant difference (P ≤ 0·05) for WT, WT/GA, eui1 and EUI-OE under the same Fe treatments.

OsYSL2 encodes an Fe-NA transporter and plays an important role in the translocation of Fe in shoots (Ishimaru et al., 2010). We monitored the expression patterns of OsYSL2 in WT, eui1 mutant and EUI-OE plants under both Fe-sufficient and Fe-deficient conditions. Iron deficiency up-regulated the expression of OsYSL2 in shoots of WT, eui1 and EUI-OE plants (Fig. 8). The expression of OsYSL2 in shoots was significantly depressed by GA application and EUI mutation under both Fe-sufficient and Fe-deficient conditions (Fig. 8). Overexpression of EUI significantly up-regulated the expression of OsYSL2 in shoots under Fe-sufficient conditions, but down-regulated its expression under Fe-deficient conditions (Fig. 8). There was an extremely significant interaction between Fe treatments and GA-related treatments in the expression of OsYSL2 of shoot (P < 0·0001).

Fig. 8.

Fig. 8.

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Quantitative RT-PCR analysis of OsYSL2 in shoots. Data are means±s.e. of three biological replicates Treatments and statistical analysis are as detailed in Fig. 7.

DISCUSSION

There is increasing evidence highlighting the involvement of GA in the mediation of abiotic stress responses (Ueguchi-Tanaka et al., 2007; Yamaguchi, 2008). However, few studies have specifically investigated the role of GA in responses of plants to deficiency in mineral nutrients. There are limited reports demonstrating the possible involvement of GA in the control of Fe homeostasis in plants. Matsuoka et al. (2014) reported that exogenous GA altered expression of Fe uptake-related genes in the ga3ox1/ga30x2 double mutant, a GA-deficient mutant of Arabidopsis under Fe-sufficient conditions. However, the authors did not evaluate the effects of GA on physiological processes associated with Fe acquisition and utilization in WT plants. In a recent study, Wild et al. (2016) provide experimental evidence showing crosstalk between GA signalling and Fe acquisition in Arabidopsis, highlighting the critical roles of GA in sensing and adapting to Fe deficiency in plants (von Wiren and Bennett, 2016). Since rice and Arabidopsis differ substantially in their responses to Fe deficiency, whether and how GA regulates Fe acquisition in rice plants remain to be evaluated. In the present study, we explored the roles of GA in the mediation of Fe acquisition under both Fe-sufficient and Fe-deficient conditions by investigating the effects of exogenous GA on growth, Fe accumulation, CHL concentration and genes involved in Fe homeostasis of WT rice seedlings. In contrast to Arabidopsis, our results revealed that an exogenous supply of GA enhanced foliar chlorosis of Fe-deficient rice seedlings, and that GA reduced CHL concentration regardless of Fe supply levels. Furthermore, we demonstrated that exogenous application of GA inhibited Fe accumulation in leaf, but not in stem and root, under both Fe-sufficient and Fe-deficient conditions. Similar to exogenous GA application, the eui1 mutant with higher endogenous bioactive GA concentration was more sensitive to Fe deficiency than WT plants, while transgenic EUI-OE rice plants were less responsive to Fe deficiency than WT plants. Another important finding in the present study is that the concentrations of biologically active GA1 and GA4 in leaves and stems of WT rice plants were significantly reduced upon exposure to Fe-deficient medium (cf. Fig. 3). Finally, we demonstrated that both exogenous application of GA and increases in endogenous GA concentration due to disruption of GA metabolism inhibited the expression of OsYSL2 in shoots (Fig. 8), which in turn may suppress the unloading of Fe from the phloem to mesophyll cells, thus resulting in enhanced foliar chlorosis. These results clearly indicate that GA plays important roles in Fe transport and translocation in strategy II rice plants.

GA is a well-known phytohormone that promotes plant growth (Yamaguchi, 2008). For instance, GA treatment can significantly increase shoot length of rice (Luo et al., 2006; Li et al., 2011). In the present study, we showed that both exogenous and endogenous GA enhancement promoted shoot length under both Fe-sufficient and Fe-deficient conditions, and that the GA synthesis inhibitor PAC and overexpression of EUI inhibited shoot length and plant biomass (Figs 1 and 4). These observations are in agreement with those reported in the literature. GA increased shoot length, but both exogenous GA application to WT and an endogenous increase of GA in the eui1 mutant enhanced Fe-deficiency-induced leaf chlorosis. Growth stimulation can increase the Fe demand of plants. In this study, exogenous application of GA enhanced shoot length under both Fe-sufficient and Fe-deficient conditions. However, GA application had no impact on shoot biomass, while leaf biomass was even reduced by GA under Fe-deficient conditions (Fig. 2). These results suggest that GA-induced leaf chlorosis may not result from high Fe demand by plants due to GA-dependent growth stimulation. Compared to WT, an endogenous increase of active GA in the eui1 mutant stimulated shoot length and biomass. Leaf Fe concentration of eui1 was significantly reduced under both Fe-sufficient and Fe-deficient conditions. Under Fe-sufficient conditions, a sufficient amount of Fe was present in the growth medium, and the reduction in foliar Fe concentration is unlikely to result from increases in Fe demand by plants. In addition to Fe, we also measured concentrations of other micronutrient and macronutrient metal ions in leaves of WT and eui1 under both Fe-sufficient and Fe-deficient conditions. Our results showed that neither exogenous nor endogenous increases in GA altered foliar Mn, Zn, Mg and K concentrations under both Fe-sufficient and Fe-deficient conditions (Supplementary Data Fig. S1). There was an increase in foliar Mn and Zn concentrations upon exposure to Fe-deficient medium. These results are in line with those reported in the literature (Morrissey and Guerinot, 2009). However, GA did not have an impact on foliar Mn and Zn concentrations regardless of Fe levels in the growth medium. These results highlight that the effect of GA is specific to Fe.

CHL is a major component of chloroplasts and has been used as an indicator of Fe status in plants (Graziano et al., 2002). There are reports showing that GA plays a role in CHL biosynthesis in Arabidopsis (Jiang et al., 2012). However, unlike Fe, the effect of GA on CHL biosynthesis may be indirect (Jiang et al., 2012). Moreover, a very high concentration of exogenous GA (100 µm) was used in the study of Jiang et al. (2012), while a much lower exogenous GA concentration (0·1 µm) was applied in the present study. Although a much higher endogenous active GA level was detected in the eui1 mutant, its CHL concentration was not affected by exogenous application of GA under Fe-sufficient conditions (Fig. 6). These results indicate that GA-enhanced leaf chlorosis may not result from its effect on CHL metabolism. In the present study, in contrast to WT, foliar Fe concentrations of WT in the presence of exogenous GA and of the eui1 mutant were reduced by 30 % under Fe-deficient conditions (Fig. 5), which may account for the aggravated foliar chlorosis and reduction in leaf biomass under Fe-deficient conditions. Correspondingly, foliar CHL and Fe concentrations were significantly increased in the dwarfed EUI-OE plants. The effect of GA on Fe concentrations of WT Arabidopsis was not determined, and shoot Fe concentration in a GA-deficient double mutant (ga3ox1/ga3ox2) was increased by 1·7-fold while exogenous application of GA reduced Fe concentration in shoot of the mutant to a level comparable to that in the WT (Matsuoka et al., 2014). However, Wild et al. (2016) reported that Fe concentration in the GA-deficient ga1-3 mutant was lower than that in WT Arabidopsis plants. The differences in Fe concentration between the GA-deficient double mutant ga3ox1/ga3ox2 and GA-deficient ga1-3 mutant may result from the sampling protocols used in the two studies. For example, in the study using the ga3ox1/ga3ox2 double mutant, shoot and root are separated, while in ga1-3 plants, the whole plant was used. In the present study, we found that exposure of WT plants to Fe-deficient medium led to a significant reduction in concentrations of GA1 and GA4, the biologically active GAs in WT plants (cf. Fig. 3). In Arabidopsis, Fe-depleted plants exhibited lower levels of bioactive GA4 and its precursors than Fe-sufficient plants (Wild et al., 2016). In maize, endogenous GA levels in shoots were reduced by Fe deficiency, but treatment with GA biosynthesis inhibitors had no impact on shoot Fe concentrations (Sekimoto et al., 2001). Different treatment times, plant species and tissues used to measure Fe concentrations in these studies may account for the different results. For example, neither Fe deficiency nor GA biosynthesis inhibitors were found to affect plant height and fresh weight, and shoot Fe concentration was determined by mixing leaves and stems (Sekimoto et al., 2001). In the present study, treatments with Fe deficiency and a GA biosynthesis inhibitor altered plant growth, and exogenous application of GA only reduced leaf Fe concentration, but it had no effect on stem Fe concentration (cf. Fig. 5), suggesting that exogenous GA possibly plays important roles in leaf Fe homeostasis. In contrast to exogenous GA application to WT plants, Fe concentration in stems was also reduced and enhanced by mutation and overexpression of EUI, respectively. This is possibly due to a difference in distribution of exogenously applied GA and biosynthetic GA of plants within leaves and stems. For exogenous application, GA was added in the hydroponic solution, and GA was taken up by roots and transported to shoots by transpiration. Since transpiration rate in leaf is higher than in stem, a greater amount of GA is expected to accumulate in leaf, leading to more accumulation of GA in leaf than in stem, and stronger responses of leaf to exogenous GA application than stem in WT plants. For eui1 and EUI-OE plants, because EUI was mainly expressed in rapidly elongating and/or dividing tissues, including the shoot apical meristems, the divisional (intercalary meristem) and elongating zones of internodes, nodes of an elongating stem and panicle (Zhu et al., 2006), GA levels in leaf and stem of the eui1 mutant and EUI-OE plants may be comparable, resulting in lower and higher Fe concentrations of leaf and stem in eui1 mutant and EUI-OE plants than WT plants, respectively. Taken together, these results indicate that GA plays important and complicated roles in Fe translocation and distribution, depending on plant species, GA level, Fe status, growth stage and different organs. In rice seedlings, leaf Fe status is likely to be negatively correlated with endogenous GA levels in plants. Supplementation of GA further aggravated leaf chlorosis in the eui1 mutant, and reduced CHL concentration of EUI-OE plants under Fe-deficient conditions, which corroborates this conclusion (Fig. 6).

In this study, we found that Fe concentration in root was significantly increased by suppression of endogenous GA regardless of Fe supply, indicating that GA is possibly involved in the regulation of Fe uptake by roots. The observations that the eui1 mutant and WT plants exhibited comparable Fe concentrations in their roots under Fe-sufficient and Fe-deficient conditions may suggest that an endogenous increase in GA level has no effect on Fe concentrations in rice roots, but exogenous application of GA significantly increased Fe concentration in roots of WT plants under Fe-sufficient conditions. In Arabidopsis, root Fe concentration in the double mutant ga3ox1/ga3ox2 was lower than in WT plant root, but Fe concentration in the double mutant was increased about 30 % in root and reduced by about 40 % in shoot in response to exogenous GA application (Matsuoka et al., 2014). These results suggest that the effect of GA on Fe concentration in roots may be more complicated than in shoots. Both exogenous GA application and endogenous inhibition of GA production by overexpression of EUI enhanced Fe concentration in rice roots, but the underlying mechanisms may be different. For EUI-OE plants, higher root Fe concentration was correlated with higher leaf and stem Fe concentration. However, for exogenous GA application, higher root Fe concentration was possibly induced by suppressing Fe transport from roots to shoots.

Iron deficiency enhances Fe uptake and translocation from roots to shoots (Vert et al., 2002). Our results imply that GA is likely to be involved in the regulation of Fe uptake, transport and translocation within rice plants. The transcription factor OsIRO2 is essential in modulating expression of genes associated with Fe homeostasis in rice plants (Kobayashi and Nishizawa, 2012). Iron deficiency induced up-regulation of OsIRO2 expression, thus activating the expression of genes responsible for Fe uptake and translocation in roots. Our results are in line with those reported in the literature. In the present study, we found that GA had no specific effects on the expression of OsIRO2 under Fe-sufficient and Fe-deficient conditions, and that the expression of genes related to Fe uptake was not in agreement with the expression pattern of OsIRO2 (Fig. 7). In Arabidopsis, exogenous application of GA to the GA-deficient Arabidopsis mutant ga3ox1/ga3ox2 up-regulated expression of Fe uptake-related genes and increased root Fe concentration, while shoot Fe concentration in the mutant was significantly reduced by GA (Matsuoka et al., 2014). These results suggest that GA possibly participates in regulating the expression of Fe-uptake-related genes at transcriptional or post-transcriptional levels, and that GA negatively regulates Fe transport from roots to shoots. Very recently, Wild et al. (2016) demonstrated that GA signalling has a tissue-specific dual function in the adaptation to a Fe-deficient environment in Arabidopsis. When Fe availability is reduced, DELLAs are accumulated in the root meristem, thereby restraining root growth and progressively excluded from epidermal cells in the root growth differentiation zone (Wild et al., 2016). The exclusion of DELLA from the site of Fe acquisition relieves FIT from DELLA-dependent inhibition, thus enhancing Fe uptake (Wild et al., 2016). In our study, whole roots were used to detect gene expression, making it impossible to monitor tissue-specific expression of genes. Wild et al. (2016) further demonstrated that the rice DELLA, SLR1, can interact with IRO2, suggesting that a similar GA-signalling mechanism may exist in strategy I Arabidopsis and strategy II rice plants in response to Fe deficiency. There have been numerous reports showing differences between Arabidopsis and rice in Fe acquisition and GA signalling. For instance, five DELLA proteins have been identified in Arabidopsis (Daviere and Achard, 2013), while only one DELLA protein, SLR1, was identified in rice (Ikeda et al., 2001). Furthermore, in Arabidopsis, constitutive FIT overexpression does not induce downstream genes, and FIT forms heterodimers with bHLH38 and/or bHLH39 under Fe-deficient conditions, and the complexes can activate the transcription of FRO2 and IRT1 in root epidermal cells (Yuan et al., 2008). In rice, no similar interacting partner has been identified, and IRO2 can directly regulate expression of various genes related to Fe utilization and genes involved in the methionine cycle in strategy II rice plants (Kobayashi and Nishizawa, 2012). Therefore, it is conceivable that GA and its signalling may have different modes to regulate Fe acquisition in rice and Arabidopsis. Further studies aiming to elucidate the molecular mechanisms by which GA modulates uptake, transport and translocation of Fe in rice plants are warranted.

OsYSL2 has proved to be involved in the phloem transport of Fe in shoots (Koike et al., 2004). Our results showed that both exogenous and endogenous increases in GA levels inhibited the expression of OsYSL2 in shoots (Fig. 8). The down-regulation of OsYSL2 may suppress the unloading of Fe from the phloem to mesophyll cells, and reduce internal Fe availability in shoots, thus resulting in enhanced leaf chlorosis. However, the inhibition by GA is around 1·5-fold under Fe-deficient conditions, and EUI-OE even depressed its expression. Since translocation of Fe within shoots remains largely to be determined (Kobayashi and Nishizawa, 2012), the depression of OsYSL2 may partly explain the phenomenon, and other mechanisms need to be explored in future studies.

CONCLUSIONS

We provide experimental evidence in support of involvement of GA in mediation of responses of strategy II rice plants to Fe deficiency. More specifically, we demonstrate that GA is involved in the modulation of Fe homeostasis in rice plants by negatively regulating Fe transport from roots to shoots, and translocation within shoots. Further studies aiming to elucidate the molecular mechanisms responsible for the effect of uptake, transport and translocation of Fe in rice plants by GA, and the associated GA-signalling transduction pathways, are warranted.

SUPPLEMENTARY DATA

Supplementary data are available online at www.aob.oxfordjournals.org and consist of the following. Table S1: the primers used for quantitative RT-PCR. Figure S1: effect of exogenous and endogenous GA increases on Mn, Zn, Mg and K concentrations of leaf under Fe-sufficient and Fe-deficient conditions.

Supplementary Material

Supplementary Data
Click here for additional data file. (105.5KB, doc)

ACKNOWLEDGEMENTS

This work was supported by the National Science Foundation of China (31672227 and 31101594) and State Key Laboratory of Vegetation and Environmental Change. We thank Professor Z. He for the gift of eui1 mutant and EUI-OE plants.

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Supplementary Data
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