Supply of sulphur to S-deficient young barley seedlings restores their capability to cope with iron shortage

Abstract

The effect of the S nutritional status on a plant's capability to cope with Fe shortage was studied in solution cultivation experiments in barley (Hordeum vulgare L. cv. Europa). Barley is a Strategy II plant and responds to Fe deficiency by secretion of chelating compounds, phytosiderophores (PS). All PS are derived from nicotianamine whose precursor is methionine. This suggests that a long-term supply of an inadequate amount of S could reduce a plant's capability to respond to Fe deficiency by limiting the rate of PS biosynthesis. The responses of barley (Hordeum vulgare L. cv. Europa) plants grown for 12 d on Fe-free nutrient solutions (NS) containing 0 or 1.2 mM SO(4)(2-), was examined after 24 h or 48 h from transfer to NS containing 1.2 mM SO(4)(2-). After the supply of S was restored to S-deprived plants, an increase in PS release in root exudates was evident after 24 h of growth in S-sufficient NS and the increment reached values up to 4-fold higher than the control 48 h after S resupply. When S was supplied to S-deficient plants, leaf ATPS (EC 2.7.7.4) and OASTL (EC 4.2.99.8) activities exhibited a progressive recovery. Furthermore, root HvST1 transcript abundance remained high for 48 h following S resupply and a significant increase in the level of root HvYS1 transcripts was also found after only 24 h of S resupply. Data support the idea that the extent to which the plant is able to cope with Fe starvation is strongly associated with its S nutritional status. In particular, our results are indicative that barley plants fully recover their capability to cope with Fe shortage after the supply of S is restored to S-deficient plants.

Fig. 1.

Shoot (A) and root (B) fresh weight of Fe-deficient barley plants. Plants were grown on S-free NS for 12 d, followed by a transfer to NS containing 1.2 mM SO₄²⁻ for 24 or 48 h. (Inserts) Response to S deficiency of barley shoots (a) and roots (b) grown on complete NS containing 1.2 mM SO₄²⁻ and 100 μM Fe(III)-EDTA (+S+Fe) or on Fe-free NS containing 0 (–S–Fe) or 1.2 mM SO₄²⁻ (+S–Fe). Data are means ±SD of four independent replications run in triplicate. Significant differences between samples are indicated by different letters (P <0.05) (n=4).

Fig. 2.

Chlorophyll content of Fe-deficient barley plants. Plants were grown on S-free NS for 12 d, followed by a transfer to NS containing 1.2 mM SO₄²⁻ (black bars) or 1.2 mM SO₄²⁻+100 μM Fe(III)-EDTA (white bars) for 24 h or 48 h. (Insert) Effect of S deficiency on leaf chlorophyll content of barley plants grown on complete NS containing 1.2 mM SO₄²⁻ and 100 μM Fe(III)-EDTA (+S+Fe) or on Fe-free NS containing 0 mM (–S–Fe) or 1.2 mM SO₄²⁻ (+S–Fe). SPAD readings were made using the first fully expanded leaf from the top of the plant. Data are means ±SD of four independent replications run in triplicate. Significant differences between samples are indicated by different letters (P <0.01) (n=4).

Fig. 3.

PSs release by Fe-deficient barley plants grown for 12 d on complete NS containing 1.2 mM SO₄²⁻ and 100 μM Fe(III)-EDTA (+S+Fe) or on Fe-free NS containing 0 (–S–Fe) (white bar) or 1.2 mM SO₄²⁻ (+S–Fe) (black bar), and by S (striped bar) or (S+Fe) resupplied plants for 48 h. Data are expressed as percentage in respect to the S-sufficient (–Fe) control (100%). Data are means ±SD of four independent replications run in triplicate. Significant differences between samples are indicated by different letters (P <0.05) (n=4).

Fig. 4.

PSs release by Fe-deficient barley plants. Plants were grown on S-free NS for 12 d, followed by a transfer to NS containing 1.2 mM SO₄²⁻ (black bars) or 1.2 mM SO₄²⁻+100 μM Fe(III)-EDTA (white bars) for 24 h or 48 h. PSs release was measured by determining the PSs content in root washings. Data are means ±SD of four independent replications run in triplicate. Significant differences between samples are indicated by different letters (P <0.01) (n=4).

Fig. 5.

Methionine content of Fe-deficient barley shoot (A) and root (B). Plants were grown on S-free NS for 12 d, followed by a transfer to NS containing 1.2 mM SO₄²⁻ (black bars) or 1.2 mM SO₄²⁻+100 μM Fe(III)-EDTA (white bars) for 24 h or 48 h. (Inserts) Response to S deficiency of barley shoots (a) and roots (b) grown on complete NS containing 1.2 mM SO₄²⁻ and 100 μM Fe(III)-EDTA (+S+Fe) or on Fe-free NS containing 0 (–S–Fe) or 1.2 mM SO₄²⁻ (+S–Fe). Data are means ±SD of four independent replications run in triplicate. Significant differences between samples are indicated by different letters (P <0.01) (n=4).

Fig. 6.

Changes in ATPS activity (nmol ATP min⁻¹ mg⁻¹ protein) in leaves (A) and roots (B) of Fe-deficient barley plants. Plants were grown on S-free NS for 12 d, followed by a transfer to NS containing 1.2 mM SO₄²⁻ (black bars) or 1.2 mM SO₄²⁻+100 μM Fe(III)-EDTA (white bars) for 24 h or 48 h. (Inserts) Effect of S deficiency on leaf and root ATPS activity of barley plants grown on complete NS containing 1.2 mM SO₄²⁻ and 100 μM Fe(III)-EDTA (+S+Fe) or on Fe-free NS containing 0 (–S–Fe) or 1.2 mM SO₄²⁻ (+S–Fe). Data are means ±SD of four independent replications run in triplicate. Significant differences between samples are indicated by different letters (P <0.05) (n=4).

Fig. 7.

Changes in OASTL activity (μmol min⁻¹ mg⁻¹ protein) in leaves (A) and roots (B) of Fe-deficient barley plants. Plants were grown on S-free NS for 12 d, followed by a transfer to NS containing 1.2 mM SO₄²⁻ (black bars) or 1.2 mM SO₄²⁻+100 μM Fe(III)-EDTA (white bars) for 24 h or 48 h. (Inserts) Effect of S deficiency on leaf and root OASTL activity of barley plants grown on complete NS containing 1.2 mM SO₄²⁻ and 100 μM Fe(III)-EDTA (+S+Fe) or on Fe-free NS containing 0 (–S–Fe) or 1.2 mM SO₄²⁻ (+S–Fe). Data are means ±SD of four independent replications run in triplicate. Significant differences between samples are indicated by different letters (P <0.05) (n=4).

Fig. 8.

RT-PCR expression analysis of a high affinity sulphate transporter (HvST1) and of an iron-phytosiderophore transporter (HvYS1) in roots of barley plants. Plants were grown on complete NS containing 1.2 mM SO₄²⁻ and 100 μM Fe(III)-EDTA (+S+Fe) or on Fe-free NS containing 0 (–S–Fe) or 1.2 mM SO₄²⁻ (+S–Fe) (A) or on S-and Fe-free NS for 12 d, followed by a transfer to NS containing 1.2 mM SO₄²⁻ (S resupply) (B) or 1.2 mM SO₄²⁻+100 μM Fe(III)-EDTA (S+Fe resupply) (C) for 24 or 48 h. A 18S PCR product was used as a cDNA calibration control.