Plants can use protein as a nitrogen source without assistance from other organisms

Abstract

Nitrogen is quantitatively the most important nutrient that plants acquire from the soil. It is well established that plant roots take up nitrogen compounds of low molecular mass, including ammonium, nitrate, and amino acids. However, in the soil of natural ecosystems, nitrogen occurs predominantly as proteins. This complex organic form of nitrogen is considered to be not directly available to plants. We examined the long-held view that plants depend on specialized symbioses with fungi (mycorrhizas) to access soil protein and studied the woody heathland plant Hakea actites and the herbaceous model plant Arabidopsis thaliana, which do not form mycorrhizas. We show that both species can use protein as a nitrogen source for growth without assistance from other organisms. We identified two mechanisms by which roots access protein. Roots exude proteolytic enzymes that digest protein at the root surface and possibly in the apoplast of the root cortex. Intact protein also was taken up into root cells most likely via endocytosis. These findings change our view of the spectrum of nitrogen sources that plants can access and challenge the current paradigm that plants rely on microbes and soil fauna for the breakdown of organic matter.

Fig. 1.

Axenic H. actites and A. thaliana use external protein for growth. (A and B) Root and shoot dry weight and nitrogen content of Hakea seedlings grown without nitrogen, with protein, or with inorganic nitrogen; 30 mg of nitrogen was supplied to each plant as protein or inorganic nitrogen (17 mM protein nitrogen as BSA or 17 mM inorganic nitrogen as NH₄NO₃). (C and D) Arabidopsis dry weight and nitrogen content grown without nitrogen or with nitrogen supplied as protein (1.5 or 6 mg BSA per ml), inorganic nitrogen (0.04 or 0.4 mg NH₄NO₃ per ml), or protein and inorganic nitrogen combined (5.4 mg BSA per ml and 0.04 mg NH₄NO₃ per ml). Bars represent averages and SD of five to eight Hakea plants and 7–10 plates with ≈80 Arabidopsis plants per plate (see also Fig. 2). Different letters indicate significant differences at P < 0.05 (Hakea) and P < 0.01 or P < 0.001 (Arabidopsis) (ANOVA, Neuman–Keuls post hoc test; data were log-transformed before analysis to account for different variances between groups).

Fig. 2.

Protein and low inorganic nitrogen in combination supported better growth of Arabidopsis than protein or low inorganic nitrogen alone. (A) No nitrogen added. (B) Protein only (6 mg BSA per ml). (C) Inorganic nitrogen only (0.04 mg NH₄NO₃ per ml). (D) Protein plus inorganic nitrogen (5.4 mg BSA per ml and 0.04 mg NH₄NO₃ per ml).

Fig. 3.

Root length of Arabidopsis increased in response to increasing protein levels in growth medium. Bars represent averages and SD of two to four plates with ≈20 Arabidopsis plants per plate. Treatments included no nitrogen added to growth medium, protein added (0.3–12 mg BSA per ml), or inorganic nitrogen (0.4 mg NH₄NO₃ per ml). Different letters indicate significant differences at P < 0.001 (ANOVA, Neuman–Keuls post hoc test).

Fig. 4.

Protease activity and protein fragments in roots of axenic Hakea and Arabidopsis after incubation with protein–chromophore complex (DQ green BSA) that fluoresces upon proteolytic degradation. (A) Protease activity resulted in fluorescence at the surface of Hakea cluster roots after 1.5 h of incubation. (B) Root cross-section of negative control with no protein–chromophore complex added. (D and F) Hakea roots incubated with protein–chromophore complex for 1.5 h (D) and 24 h (F) and cross-sectioned. (H and J) Arabidopsis roots incubated for 6 h. (C, E, G, and I) Bright-field images of D, F, H, and J, respectively. Images in B, E, and F were taken with a fluorescence microscope; all others were taken with a confocal microscope.

Fig. 5.

Proteolysis and/or depletion of BSA as observed by analyzing protein solution incubated with H. actites (A) and A. thaliana (B) roots. The presence of protein in the incubation solution of intact Hakea roots is strongly reduced over the course of 3.5 h. No peptides of lower molecular mass were observed in the incubation solution of Hakea. The incubation solution of Arabidopsis roots contained decreasing amounts of BSA but also a smaller protein fragment that increased over time. Protein solution was supplied to the root as 50 μg BSA per ml. Numbers on the left side identify peptide standards of 15–116 kDa (BSA has a molecular mass of 66 kDa); numbers across columns indicate hours of incubation.

Fig. 6.

Roots of axenic Hakea and Arabidopsis seedlings incubated with GFP show intact protein associated with root surfaces, root hairs, and root cortex cells. (A) GFP was associated with Hakea root hairs. (B) The whole root surface of Arabidopsis. GFP also was observed inside Arabidopsis root hairs and cortex cells. (C, F, and I) SI Movie 1 shows cytoplasmic streaming in C. (E and H) Bright-field images of D and G, respectively, and combined in F and I. Fluorescent images were taken with a confocal microscope.

Fig. 7.

EM of Hakea root-transverse sections show GFP in apoplast and root cortex cells. (A and B) Roots were incubated without GFP (A, negative control) and with GFP (B) and probed with immunogold labeled anti-GFP. GFP was detected in the apoplast, cell wall (cw), and cytoplasm (c) of root cortex cells. Gold labeling is marked with short arrows. The plasma membrane (pm) also is indicated. (Scale bars: 1 μm.)