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First published online 23 January 2007
doi: 10.1242/jcs.001362

Journal of Cell Science 120, 596-605 (2007)
Published by The Company of Biologists 2007
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Targeted alterations in iron homeostasis underlie plant defense responses

Guosheng Liu1,*, David L. Greenshields1,*, Ramaswami Sammynaiken2, Rozina N. Hirji3, Gopalan Selvaraj3 and Yangdou Wei1,{ddagger}

1 Department of Biology, University of Saskatchewan, 112 Science Place, Saskatoon, SK S7N 5E2, Canada
2 Saskatchewan Structural Sciences Center, University of Saskatchewan, 110 Science Place, Saskatoon, SK S7N 5C9, Canada
3 Plant Biotechnology Institute, National Research Council of Canada, 110 Gymnasium Place, Saskatoon, SK S7N 0W9, Canada


Figure 1
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  		Fig. 1. Targeted Fe redistribution in wheat leaves after Bgt attack. (A) Fe concentration increases in Bgt-attacked tissue. Metal concentrations determined by ICPMS analysis of wheat leaf epidermal cells and underlying mesophyll cells in response to Bgt attack, 24 hpi (filled bars) or before inoculation (open bars). The mean values (± s.d.) of three independent treatments are shown. Asterisks indicate significant difference (P<0.01) before and after inoculation, based on Student's t-test. (B) In situ Fe2+ Prussian Blue staining of wheat epidermis 24 hpi with Bgt. (C) In situ Fe3+ Prussian Blue staining of wheat epidermis 24 hpi with Bgt. (D) In situ Fe3+ Prussian Blue staining of epidermal peels 24 hpi with Bgt in corn, barley, oat, sorghum and millet. (E) The actin filament disruptor cytochalasin A (cytA) blocks Fe accumulation at CWAs. Graph shows the reduction in Fe3+ accumulation at agt-associated CWAs (means ± s.d., n=300 based on 100 Bgt attack sites per leaf on three leaves). agt, appressorial germ tube; c, conidium; ha, halo; gc, guard cell; N, epidermal nucleus; n, fungal nucleus; pa, papilla; pgt, primary germ tube; tri, trichome. Scale bar, 20 µm.

 

Figure 2
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  		Fig. 2. Chelatable, redox-active Fe accumulation in wheat leaves after Bgt attack. (A) Wheat epidermis 24 hpi with Bgt stained for Fe3+ after pretreatment with the Fe3+ chelator DFO. (B) In situ calcein staining of Fe3+ in wheat leaves 24 hpi with Bgt before (left panel) and after (right panel) treatment with the Fe chelator DFO. (C) Wide-range X-band EPR spectrometry of wheat leaves 24 hpi with (blue solid line) or without (black broken line) Bgt inoculation. Insets are the enlarged high-resolution scanning for high-spin and low-spin Fe3+, respectively. The g values of high-spin Fe3+ are indicated. Spectral intensities were normalized relative to sample amount. Two independent experiments showed similar results. agt, appressorial germ tube; c, conidium; ha, halo; gc, guard cell; N, epidermal nucleus; pa, papilla. Scale bar, 20 µm.

 

Figure 3
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  		Fig. 3. Fe accumulation mediates DAB oxidation at CWAs. (A) H2O2 accumulation in CWAs revealed by DAB staining 24 hpi. (B) Double staining for H2O2 and Fe3+ (blue) 24 hpi. (C) Double staining for H2O2 and Fe3+ at a primary germ tube-associated CWA 5 hpi. (D) Vesicle-like bodies within a CWA double stained for H2O2 and Fe3+ 24 hpi. (E) Wheat leaves with (right) or without (left) pretreatment of 1.5 mM DFO were stained using DAB and photographed 24 hpi. (F-H) DFO blocks H2O2 generation at CWAs. Upper panel: without DFO pretreatment. Lower panel: with 1.5 mM DFO pretreatment. (F) DFO blocks H2O2 generation at appressorial germ tube-associated CWAs. (G) DFO blocks H2O2 generation at primary germ tube-associated CWAs but not cytoplasmic aggregation (arrowhead). (H) Pretreatment with DFO does not affect nuclear migration (arrows) at CWAs. agt, appressorial germ tube; c, conidium; N, host nucleus; pgt, primary germ tube. Scale bar, 20 µm.

 

Figure 4
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  		Fig. 4. Fe overload causes apoplastic H2O2 production and induces Fe homeostasis- and defense-related gene expression. (A) ICPMS analysis of Fe3+-EDTA uptake in wheat leaves. Values represent mean ± s.d. (n=3). (B) DAB staining for H2O2 in wheat leaves 12 hours after treatment with either Fe3+-EDTA (middle and right panels) or EDTA (Mock, left panel). (C) Northern blot analysis of time-dependent induction of Fe homeostasis- and defense-related genes following loading with 500 µM EDTA (Mock) or 500 µM Fe3+-EDTA at the time points indicated. Even loading of total RNA was monitored by ethidium bromide staining of rRNA. (D) Northern blot analysis of Fe3+-EDTA concentration-dependent gene expression. (E) Northern blot analysis of Fe3+-dependent and H2O2-dependent gene expression.

 

Figure 5
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  		Fig. 5. Pathogen attack promotes cytosolic Fe depletion, leading to defense gene expression. (A) RT-PCR of mRNAs from epidermal (e) and mesophyll (m) tissues of wheat leaves before (0 hpi) and after (24 hpi) inoculation with Bgt. TmGPD1 was used to check equal mRNA usage. (B) Northern blot analysis of TmNAS1 and TmPR1b expression in wheat leaves 0-72 hpi with Bgt. (C) Northern blot analysis of total RNA from wheat leaves treated with either DFO alone or DFO followed by H2O2 for either 6 or 18 hours.

 

Figure 6
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  		Fig. 6. H2O2 promotes cytosolic Fe depletion and Fe efflux. (A,B) Confocal imaging of Fe depletion in wheat suspension cells, based on calcein-AM fluorescence. Images (lower panels) depict the fluorescence at representative time points. Scale bar, 20 µm. Time course of calcein-AM uptake (A); H2O2-mediated cytosolic Fe depletion (B). (C,D) H2O2-induced Fe efflux in wheat suspension cells. Bar values represent mean ± s.d. (n=3). Cells were treated with glucose/glucose oxidase (G/GO) (5 mM/5 U ml–1) or H2O2 (10 mM), with or without catalase (Cat) (10 U ml–1) or vanadate (NaVO3) (100 µM), respectively (C); cells treated with 10 mM H2O2 and 20 or 200 µM cycloheximide (CHX) (D).

 

Figure 7
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  		Fig. 7. The proposed role of Fe homeostasis in plant defense responses. Pathogen attack elicits the targeted Fe redistribution to the apoplast, which leads to Fe deficiency in the cytosol of attacked cells. The accumulated apoplastic Fe mediates the oxidative burst, which further stimulates Fe efflux and intracellular Fe deficiency. H2O2 and Fe deficiency induce expression of defense-related genes while suppressing the expression of Fe storage-related genes.

 

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© The Company of Biologists Ltd 2007