Modulating tiller formation in cereal crops by the signalling function of fertilizer nitrogen forms

Abstract

Cereal crop yield comprises interrelated components, among which the number of tillers is highly responsive to nitrogen fertilization. We addressed the hypothesis of whether the supply of different nitrogen forms can be employed to manipulate the tiller number in cereal crops. Relative to urea or ammonium, exclusive supply of nitrate increased tiller number in hydroponically-grown barley plants. Thereby, tiller number correlated positively with the root-to-shoot translocation rate of endogenous cytokinins. External supply of a synthetic cytokinin analog further stimulated tillering in nitrate-containing but not in urea-containing nutrient solution. When the cytokinin analog 6-benzylaminopurine riboside was externally supplied to roots, its translocation to shoots was 2.5 times higher in presence of nitrate than in presence of urea or ammonium, suggesting that cytokinin loading into the xylem is affected by different nitrogen forms. We then translated this finding to field scale, cultivated winter wheat in four environments, and confirmed that nitrate fertilization significantly increased tiller number in a dose-dependent manner. As assessed in 22 winter wheat cultivars, nitrogen form-dependent tiller formation was subject to substantial genotypic variation. We conclude that cytokinin-mediated signaling effects of fertilizer nitrogen forms can be employed as a management tool to regulate the tiller number in cereal crops.

Figure 1

Dry weight, tiller number and cytokinin translocation rates in barley plants as affected by the supply of different nitrogen forms. (A) Dry weight per plant, (B) tiller number per plant incl. the primary shoot axis, (C) dry weight per single tiller, (D) root-to-shoot translocation rate of the cytokinin forms zeatin (Z) and zeatin-riboside (ZR) and (E) root-to-shoot translocation rate of the cytokinin precursors isopentenyl-adenine (i-Ade) and isopentenyl-adenosine (i-Ado) as determined in the xylem bleeding sap. Five days-old barley seedlings were transferred for 40 days to nutrient solution containing the following N sources: 0.5 mM KNO₃, 0.38 mM KNO₃ + 0.06 mM urea (75:25% N), 0.25 mM KNO₃ + 0.13 mM urea (50:50% N), 0.12 mM KNO₃ + 0.19 mM urea (25:75% N), 0.25 mM urea, 0.25 mM NH₄NO₃. The pH was buffered at pH 6.6 by Ca(HCO₃)₂, and wherever indicated 75 µg L⁻¹ of the urease inhibitor PPD was added. Bars represent means ± SD, n = 3 biological replicates. Different letters indicate significant differences among treatments at p < 0.05 by Tukey’s test.

Figure 2

Growth and tiller formation in barley after external supplementation of cytokinins in the presence of nitrate or urea. (A) Dry weight of shoots and roots and (B) tiller number of spring barley plants precultured hydroponically on ammonium nitrate before transfer to different N sources: 0.5 mM KNO₃, 0.38 mM KNO₃ + 0.06 mM urea (75:25% N), or 0.12 mM KNO₃ + 0.19 mM urea (25:75% N). The pH was buffered at pH 6.6 by Ca(HCO₃)₂. The cytokinin analogue 6-benzylaminopurine (BA) was supplied at a concentration of 10 µM at the beginning of tillering (3-leaf stage). Tillers were counted at the 7-leaf stage. The urease inhibitor PPD was supplemented at 75 µg L⁻¹. Bars represent means ± SD, n = 20 biological replicates. Different letters indicate significant differences among treatments at p < 0.05 by Tukey’s test.

Figure 3

Translocation rate of benzylaminopurine riboside (BAR) and potassium in the xylem sap of barley plants as affected by the presence of different nitrogen forms. (A) Benzyl-aminopurine translocation rate and (B) potassium translocation rate as determined in the xylem bleeding sap from spring barley precultured hydroponically until the 5-leaf stage on ammonium nitrate, then for 24 h on N-free nutrient solution and then for 48 h on pH-buffered 0.5 mM potassium nitrate, urea or ammonium sulfate. BAR was supplied at a concentration of 10 µM 12 h before collection of the xylem bleeding sap. Bars represent means ± SD, n = 3 biological replicates. Different letters indicate significant differences among treatments at p < 0.05 by Tukey’s test.

Figure 4

The influence of fertilizer N forms on tiller formation of field-grown winter wheat. Plants were grown either on the site Langenstein (A,C) or Dörrhof (B,D) in the growth season 2005/2006 (A,B) or 2006/2007 (C,D). Tiller density was determined at the end of vegetative development (BBCH 31) of winter wheat supplemented with 40 or 80 kg N ha⁻¹ as starter dressing in 2006 and 30 or 60 kg N ha⁻¹ as starter dressing in 2007 in the form of urea plus urease inhibitor (UI) and nitrification inhibitor (NI), providing urea-dominated N supply, urea plus nitrification inhibitor (ammonium-dominated N supply), or ammonium nitrate (nitrate-dominated N supply). Bars represent means ± SD, n = 4 independent replicate plots. Different letters indicate significant differences among fertilizer treatments at p < 0.05 by Tukey’s test.

Figure 5

Genotypic variation of tiller formation in 22 field-grown winter wheat cultivars in dependence of the form of N nutrition. Tiller density was determined at the end of vegetative development (BBCH 31) of winter wheat plants supplemented with 80 kg N ha⁻¹ as starter dressing in the form of urea plus urease inhibitor (UI) and nitrification inhibitor (NI), providing urea-dominated N supply, or ammonium nitrate (nitrate-dominated N supply). Plants were grown on the site Gatersleben in 2009/2010. Bars represent means ± SD, n = 4 independent replicate plots. Asterisks indicate significant differences among fertilizer treatments at p < 0.05 by Tukey’s test.