Effects of phosphorus and nitrogen on nodulation are seen already at the stage of early cortical cell divisions in Alnus incana

Abstract

Background and aims: The present work aimed to study early stages of nodulation in a chronological sequence and to study phosphorus and nitrogen effects on early stages of nodulation in Alnus incana infected by Frankia. A method was developed to quantify early nodulation stages in intact root systems in the root hair-infected actinorhizal plant A. incana. Plant tissue responses were followed every 2 d until 14 d after inoculation. Cortical cell divisions were already seen 2 d after inoculation with Frankia. Cortical cell division areas, prenodules, nodule primordia and emerging nodules were quantified as host responses to infection.

Methods: Seedlings were grown in pouches and received different levels of phosphorus and nitrogen. Four levels of phosphorus (from 0.03 to 1 mM P) and two levels of nitrogen (0.71 and 6.45 mM N) were used to study P and N effects on these early stages of nodule development.

Key results: P at a medium concentration (0.1 mM) stimulated cell divisions in the cortex and a number of prenodules, nodule primordia and emerging nodules as compared with higher or lower P levels. A high N level inhibited early cell divisions in the cortex, and this was particularly evident when the length of cell division areas and presence of the nodulation stages were related to root length.

Conclusions: Extended cortical cell division areas were found that have not been previously shown in A. incana. The results show that effects of P and N are already expressed at the stage when the first cortical cell divisions are induced by Frankia.

Fig. 1.

Early stages of nodule formation in A. incana inoculated with Frankia. (A) Non-inoculated root showing straight root hairs (arrowheads). (B) 2 DAI. CCDA (stars) was limited to a few cells in one cell layer; RHD (arrowhead) and xylem (X). (C) 4 DAI. CCDA (stars) is enlarged and takes place in two cell layers; RHD (arrowheads) and xylem (X). (D) 6 DAI. CCDA (stars) is now occurring in three cell layers and cell size is increasing; RHD (arrowheads). (E) 8 DAI. Vascular bundles (arrowheads) are developing in a nodule primordium. (F) 10 DAI. Root showing root hair deformation and underneath at least two nodule primordia (stars); RHD (arrowhead). In (A–F), the scale bar is 10 μm. (G) Lateral root primordium occupying a narrow part of the cortex. There is no CCDA near the lateral root primordium. Scale bar = 20 μm.

Fig. 2.

Total length of CCDA per plant. Each bar represents the mean ± s.e. for 15 plants. The letters ‘l’, ‘mh’ and ‘h’ stand for low, moderately high and high levels of P and N in the nutrient solution as defined in Table 1. Different letters on top of the bars denote a statistically significant difference.

Fig. 3.

Number of prenodules, nodule primordia and emerging nodules combined per plant. Each bar represents the mean ± s.e. for 15 plants. The letters ‘l’, ‘mh’ and ‘h’ stand for low, moderately high and high levels of P and N in the nutrient solution as defined in Table 1. Different letters on top of the bars denote a statistically significant difference.

Fig. 4.

Number of prenodules, nodule primordia and emerging nodules combined in relation to total length of the CCDA. Each bar represents the mean ± s.e. for 15 plants. The letters ‘l’, ‘mh’ and ‘h’ stand for low, moderately high and high levels of P and N in the nutrient solution as defined in Table 1. Different letters on top of the bars denote a statistically significant difference.

Fig. 5.

Root length per plant. Each bar represents the mean ± s.e. for 15 plants. The letters ‘l’, ‘mh’ and ‘h’ stand for low, moderately high and high levels of P and N in the nutrient solution as defined in Table 1. Different letters on top of the bars denote a statistically significant difference.

Fig. 6.

Total length of the CCDA in relation to root length. Each bar represents the mean ± s.e. for 15 plants. The letters ‘l’, ‘mh’ and ‘h’ stand for low, moderately high and high levels of P and N in the nutrient solution as defined in Table 1. Different letters on top of the bars denote a statistically significant difference.

Fig. 7.

Number of prenodules, nodule primordia and emerging nodules combined per root length. Each bar represents the mean ± s.e. for 15 plants. The letters ‘l’, ‘mh’ and ‘h’ stand for low, moderately high and high levels of P and N in the nutrient solution as defined in Table 1. Different letters on top of the bars denote a statistically significant difference.