Nitric oxide is the shared signalling molecule in phosphorus- and iron-deficiency-induced formation of cluster roots in white lupin (Lupinus albus)

Abstract

Background and aims: Formation of cluster roots is one of the most specific root adaptations to nutrient deficiency. In white lupin (Lupinus albus), cluster roots can be induced by phosphorus (P) or iron (Fe) deficiency. The aim of the present work was to investigate the potential shared signalling pathway in P- and Fe-deficiency-induced cluster root formation.

Methods: Measurements were made of the internal concentration of nutrients, levels of nitric oxide (NO), citrate exudation and expression of some specific genes under four P × Fe combinations, namely (1) 50 µm P and 10 µm Fe (+P + Fe); (2) 0 P and 10 µm Fe (-P + Fe); (3) 50 µm P and 0 Fe (+P-Fe); and (4) 0 P and 0 Fe (-P-Fe), and these were examined in relation to the formation of cluster roots.

Key results: The deficiency of P, Fe or both increased the cluster root number and cluster zones. It also enhanced NO accumulation in pericycle cells and rootlet primordia at various stages of cluster root development. The formation of cluster roots and rootlet primordia, together with the expression of LaSCR1 and LaSCR2 which is crucial in cluster root formation, were induced by the exogenous NO donor S-nitrosoglutathione (GSNO) under the +P + Fe condition, but were inhibited by the NO-specific endogenous scavenger 2-(4-carboxyphenyl)-4, 4, 5, 5-tetramethylimidazoline-1-oxyl- 3-oxide (cPTIO) under -P + Fe, +P-Fe and -P-Fe conditions. However, cluster roots induced by an exogenous supply of the NO donor did not secrete citrate, unlike those formed under -P or -Fe conditions.

Conclusions: NO plays an important role in the shared signalling pathway of the P- and Fe-deficiency-induced formation of cluster roots in white lupin.

Fig. 1.

Cluster root (CR) morphology and cluster root number per white lupin plant grown with different concentrations of P and Fe. After germination, white lupin plants were cultured in four different growth regimes (+P + Fe, –P + Fe, +P–Fe and –P–Fe) for 20 d. (A) Number of cluster roots per plant in the four treatments. (B) Total length of the cluster root zone per plant. (C) Average length of rootlets. (D) Representative cluster root segments of white lupin grown under the four treatments. The circles indicate the primordium zone. (E) The primordium zone of each cluster root was stained with methylene blue and observed under a stereoscope, zoomed in on the encircled part of (D). Vertical bars indicate ± s.e. (n = 8). Different letters in the graphs indicate significantly different values at the 5 % level. Scale bars = 1 cm.

Fig. 2.

Concentrations of (A) inorganic P (Pi) and (B) soluble Fe in shoots, and (C) total P and (D) Fe in shoots and roots of white lupin grown at different concentrations of P and Fe. After germination, plants were grown in four different growth regimes (+P + Fe, –P + Fe, +P–Fe and –P–Fe) for 20 d. Vertical bars indicate ± s.e. (n = 8). Different letters in the graphs indicate significantly different values at the 5 % level.

Fig. 3.

Localization of NO in cluster roots of white lupin grown at different concentrations of P and Fe. Plants were grown in four different growth regimes (+P + Fe, –P + Fe, +P–Fe and –P–Fe) for 20 d. Segments of cluster roots were excised and stained by DAF-FM DA. Cross-sections of the cluster root primordium zone were obtained and observed by epifluorescence microscopy. (A) A cluster root primordium initiated from pericycle cells. (B) Radial growth of a rootlet primordium. (C) A rootlet emerged from the epidermis. +P + Fe: cross-section of a cluster root segment from a plant grown at +P + Fe. Magnification: ×10.

Fig. 4.

Effect of an NO donor (GSNO) and scavenger (cPTIO) on the number of cluster roots (CR) of white lupin grown at different concentrations of P and Fe. Plants were cultured in four different growth regimes (+P + Fe, –P + Fe, +P–Fe and –P–Fe) for 14 d, and then supplemented with 0–200 µm GSNO (A) for a further 6 d, or transferred to 100 mL of nutrient solution with 0–500 µm cPTIO (B) for a further 6 d. Vertical bars indicate ± s.e. (n = 6).

Fig. 5.

Effect of an NO donor (GSNO) and scavenger (cPTIO) on relative gene expression of LaSCR1 and LaSCR2 in non-cluster roots of white lupin. Plants were grown in +P + Fe conditions for 20 d, and then supplemented with 200 µm GSNO or 500 µm cPTIO for a maximum treatment of 24 h. Data are means ± s.e. (n = 3).

Fig. 6.

Effect of an NO donor (GSNO) and scavenger (cPTIO) on relative gene expression of LaSCR1 and LaSCR2 in primordia zones of white lupin. Plants were grown in four different growth regimes (+P + Fe, –P + Fe, +P–Fe and –P–Fe) for 20 d, and then supplemented with 0–200 µM GSNO or 500 µm cPTIO for a further 6 h. Data are means ± s.e. (n = 3).

Fig. 7.

Gene expression in cluster roots (CR) of white lupin grown at different concentrations of P and Fe. After germination, plants were grown in four different growth regimes (+P + Fe,–P + Fe, +P–Fe and –P–Fe) for 20 d. (A) LaPEPC3. (B) LaMATE. Data are means ± s.e. (n = 3).

Fig. 8.

Citrate exudation in cluster roots induced by GSNO or different concentrations of P and Fe. Plants were grown in four different growth regimes (+P + Fe, –P + Fe, +P–Fe and –P–Fe) for 14 d, and then supplemented with 0–200 µm GSNO for a further 6 d in the +P + Fe treatment. Vertical bars indicate ± s.e. (n = 8). Different letters indicate significantly different values at the 5 % level.