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

Mineral nitrogen fertilizers are applied in various chemical forms (urea, nitrate, ammonium), which differ in soil behavior, environmental impacts, plant uptake, assimilation sites, and downstream effects on plant physiology and development. Prior work shows ammonium can reduce photosynthesis and water use yet increase drought and pathogen tolerance, while nitrate tends to promote vegetative growth and delay senescence. Under controlled conditions, contrasting N forms strongly affect plant development, though field effects are often weaker due to microbial interconversion of N forms. Tiller number is a key, N-responsive yield component in cereals, influencing grain-to-straw ratio and resource use efficiency; it is modulated by species/cultivar, density, light, and climate, and often declines via tiller abortion. Physiologically, N regulates hormones: N deficiency promotes strigolactones that suppress branching, whereas nitrate stimulates cytokinin biosynthesis and root-to-shoot translocation. However, the impact of ammonium or urea on cytokinin translocation and tillering in cereals has been inconsistent. The study tests whether manipulating the supplied N form (nitrate vs reduced forms) can reliably modulate tiller number via cytokinin signaling in hydroponics and field conditions.

• Agronomic choice of N form considers availability, cost, accompanying ions, and environmental impacts (mobility, pH effects, leaching, denitrification, volatilization). Urease and nitrification inhibitors can partly stabilize urea and ammonium but do not fully prevent conversion.
• Physiological comparisons show ammonium versus nitrate nutrition exerts opposing effects on photosynthesis, respiration, water use, stress tolerance, and development. Exclusive ammonium can accelerate flowering; nitrate promotes vegetative growth and delays senescence.
• Mixed N sources (ammonium nitrate) have been reported to enhance wheat tillering more than single forms.
• Hormonal basis: N deficiency elevates strigolactones (suppress branching); nitrate increases cytokinin biosynthesis and translocation. Prior studies in tobacco and tomato showed nitrate-stimulated cytokinin translocation; ammonium can depress cytokinin xylem transport.
• In wheat, relations between N forms, cytokinins, and tillering were previously contradictory, likely due to soil pH effects and unaccounted N-form conversion.
• Molecular links: nitrate-inducible IPT genes for cytokinin biosynthesis; side-chain modification (trans-zeatin formation via P450 enzymes) is critical for shoot growth; ABCG14 mediates cytokinin xylem loading in Arabidopsis.

Hydroponic barley experiments:

• Plant material: Summer barley (cv. Henni) germinated 4 days in CaSO4-saturated quartz sand.
• Growth conditions: Nutrient solution with macro/micronutrients; pH 6.6 buffered by Ca(HCO3)2; 16/8 h light/dark, 280 µmol m−2 s−1, 25/20 °C, 60% RH.
• N treatments: 0.5 mM total N supplied as nitrate (KNO3), ammonium (NH4NO3 or (NH4)2SO4), urea, or mixtures (e.g., nitrate:urea at 75:25, 50:50, 25:75% N). To prevent exogenous urea hydrolysis, 75 µg L−1 phenylphosphorodiamidate (PPD) was added; controls confirmed PPD had no growth effect.
• Duration and measurements: Five-day-old seedlings transferred for 40 days; tillers counted at 7-leaf stage; shoots/roots harvested, freeze-dried, and weighed; tissue ammonium and urea quantified (HPLC with OPA derivatization for NH4+, colorimetric diacetylmonoxime assay for urea). Dry weight per single tiller measured.

Cytokinin translocation assays:

• Xylem bleeding sap collection: Shoots decapitated ~10 mm above hypocotyl; silicone tube affixed; sap collected 4 h, with 0.5 mM K2SO4 added to boost root pressure; sap from ~20 plants pooled per biological replicate (n=3).
• Cytokinin quantification: PVPP cleanup, C18 Sep-Pak fractionation; radioimmunoassay detection of zeatin (Z), zeatin riboside (ZR), isopentenyl-adenine (i-Ade), and isopentenyl-adenosine (i-Ado). K concentration by flame photometry.

Cytokinin supplementation studies:

• Long-term BA/BAR supplementation: Plants precultured on 0.25 mM NH4NO3 to 3-leaf stage, then transferred to 0.5 mM KNO3, or nitrate:urea mixes (75:25% N; 25:75% N). 10 µM BA applied at start of tillering; in some tests, BAR also applied. Growth to 7-leaf stage; dry weights and tiller numbers measured (n up to 20 plants per treatment). PPD included.
• Short-term BAR transport: Plants at 5-leaf stage preconditioned 24 h N-free, then 48 h on 0.5 mM nitrate (KNO3), urea, or ammonium ((NH4)2SO4). 10 µM BAR added 12 h before sap collection. BAR in xylem quantified by ELISA with specific antibody; potassium translocation rate assessed to account for exudation differences.

Field trials (winter wheat):

• Sites and seasons: Langenstein (loamy silt, NE Germany; cultivar Cubus) and Dörrhof (loamy clay, S Germany; cultivar Schamane) in 2005/2006 and 2006/2007; additional cultivar panel at Gatersleben in 2009/2010.
• Basal fertilization: October sowing with 30 kg N ha−1 as ammonium sulfate.
• Starter dressing (March): Compared (i) fully stabilized urea (urease inhibitor NBPT + nitrification inhibitor), urea-dominated; (ii) urea + nitrification inhibitor, ammonium-dominated; (iii) ammonium nitrate, nitrate-dominated (rapid NH4+ adsorption and nitrification). Doses: 40 or 80 kg N ha−1 (2006), 30 or 60 kg N ha−1 (2007). Later topdressings followed local practice (e.g., additional ammonium nitrate at BBCH 31/32 and BBCH 39).
• Measurements: Tiller density at BBCH 31/32; yield components and grain yield at harvest. Design: fully randomized block with four replicates per treatment. Cultivar panel: 22 elite winter wheat cultivars fertilized with 80 kg N ha−1 as NBPT-coated urea or ammonium nitrate; tiller density at BBCH 31.
• Statistics: Tukey’s test at p<0.05 for treatment comparisons; data presented as mean ± SD with n specified per experiment.

Hydroponic barley:

• Exclusive nitrate (0.5 mM) produced the highest shoot biomass and on average 2.7 tillers per plant; stepwise replacement by urea decreased dry weight and tiller number. Pure urea-fed plants formed only a single short main shoot with no tillers.
• Urease inhibitor PPD itself did not affect growth or tillering.
• Tissue ammonium/urea levels increased with urea supply but did not align with growth depression; absence of toxicity symptoms and higher individual tiller dry weights at 75% urea-N indicate toxicity was unlikely the main cause.
• Cytokinin translocation: Zeatin and zeatin-riboside translocation rates in xylem sap decreased strongly with increasing urea-N; isopentenyl-type cytokinin translocation was much less affected, indicating a specific impact on active cytokinin forms rather than sap volume alone.
• Cytokinin supplementation: Exogenous BA or BAR increased tiller number only under exclusive nitrate supply; no tiller stimulation occurred in 25% or 75% urea-N treatments despite similar BA effects on biomass reduction across N forms.
• BAR transport assay: Root-to-shoot translocation of exogenous BAR was ~2.5-fold higher with nitrate than with ammonium or urea; potassium translocation rates did not differ by N form, supporting a nitrate-specific stimulation of cytokinin xylem loading.

Field winter wheat:

• Across four environment-by-dose combinations, nitrate-dominated starter dressing (ammonium nitrate) significantly increased tiller density relative to unfertilized controls and was generally more effective than fully stabilized urea. Differences ranged from about 14 to 140 tillers m−2 in favor of nitrate depending on site and dose.
• Dose-dependence: Stronger effects at higher N doses and on less fertile soil (Dörrhof), consistent with higher reliance on fertilizer-derived N versus mineralized soil N.
• Yield consequences were environment-dependent: In a dry spring (2005/2006), higher tiller numbers with nitrate did not translate to higher yield due to tiller abortion; stabilized urea sometimes yielded better via reduced stand density and water use. In 2006/2007 at Dörrhof, higher nitrate-induced tiller density translated into higher grain yield under favorable grain filling.
• Genotypic variation: Among 22 cultivars, 11 showed significantly higher tiller numbers under nitrate-dominated fertilization; most others showed the same trend. Genotypic range spanned ~200 tillers m−2 at BBCH 31, and cultivar rankings differed between N forms, indicating genetic control of N-form responsiveness.

Overall: Three converging lines of evidence support that nitrate enhances cytokinin root-to-shoot translocation and tillering, whereas urea/ammonium suppress it, and that fertilizer N form can be used to modulate tiller number in cereals.

The study addressed whether fertilizer N form can manipulate tiller number via hormonal signaling. Results demonstrate that nitrate, compared with urea or ammonium, elevates root-to-shoot translocation of active cytokinins (zeatin/ZR and exogenous BAR), aligning with enhanced tiller formation in barley and higher tiller densities in field-grown wheat. Exogenous cytokinin promoted tillering only in nitrate-fed plants, pointing to N-form effects on cytokinin xylem loading rather than uptake or general growth. These findings link agronomic N-form choice to developmental regulation through cytokinin signaling. Mechanistically, nitrate likely stimulates both cytokinin biosynthesis and especially xylem loading. The weak response of isopentenyl-type cytokinins supports the importance of trans-zeatin side-chain modification (P450-mediated transhydroxylation) for active cytokinin pools affecting shoot branching. N-form regulation of cytokinin transporters (e.g., ABCG14 homologs) may underlie enhanced loading under nitrate. In the field, the extent to which nitrate-dominated fertilization increases tiller density depends on soil fertility, climate (particularly moisture during tillering and grain filling), and cultivar genetics. Because tiller abortion and sink limitations can offset gains in tiller number, optimizing N form becomes a tool for tailoring stand density to expected environmental conditions, potentially improving resource use and yield stability. The genotype-by-N-form interactions suggest breeding opportunities for cultivars with favorable tillering responses to specific N forms.

• Main contributions: Demonstrated across hydroponic barley and field-grown wheat that nitrate, relative to urea or ammonium, enhances tiller formation by promoting root-to-shoot translocation of active cytokinins. Established that exogenous cytokinin increases tillers only under nitrate, implicating N-form control over cytokinin xylem loading. Showed dose-dependent field responses and substantial genotypic variation in N-form-responsive tillering.
• Practical implication: Fertilizer N form can be used as a management lever to adjust tiller number. Use nitrate-based starter dressings to boost tillering after poor tiller establishment (e.g., late sowing, harsh winters). Use stabilized urea or ammonium-dominated N to restrain excessive tillering (e.g., mild winters, early sowing), potentially benefiting yield under dry generative phases.
• Future research: Identify and characterize cereal homologs of cytokinin xylem exporters (e.g., ABCG14) and regulatory networks modulating their expression by N forms; assess the role of cytochrome P450 enzymes in trans-zeatin biosynthesis under different N forms; dissect genotype-by-N-form interactions and map loci controlling differential tillering responses; evaluate long-term yield stability and environmental outcomes of N-form-based tiller management across diverse environments.

• Field N forms undergo microbial interconversion despite inhibitor use, reducing treatment distinctness; thus, observed effects may be conservative estimates of pure-form impacts.
• Environmental dependence: Climate strongly influenced translation of tiller number to yield via tiller abortion, limiting generalizability to yield outcomes.
• Hydroponic conditions included buffered pH and stabilized urea; soil pH dynamics and rhizosphere interactions in real fields may alter hormone and N-form effects.
• Cytokinin transport inferences are based on xylem bleeding sap and exogenous analog assays; direct measurements of transporter expression/activity and endogenous cytokinin fluxes in situ were not performed.
• Genotype panel size (22 cultivars) and single-site/year evaluation for G×N-form interaction limits broader genetic inference.
• Sample sizes in some physiological assays were modest (e.g., n=3 biological replicates for sap), though supported by consistent trends across experiments.