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

Cereal crop yield is a complex trait influenced by several interconnected components, with tiller number being highly responsive to nitrogen (N) fertilization. Different forms of mineral N fertilizers (urea, nitrate, ammonium) are available, each with distinct agronomic properties and environmental impacts. While the choice of N form often prioritizes commercial factors and environmental considerations (soil mobility, pH effects, leaching, denitrification, ammonia volatilization), their physiological effects on plants are significant. Ammonium and nitrate, for example, differ in their uptake, translocation rates, assimilation sites, and impact on primary and secondary metabolism, affecting plant growth and development. Ammonium can decrease photosynthesis and respiration but enhance drought and pathogen tolerance, while nitrate stimulates vegetative growth and delays senescence. These effects are often more pronounced under controlled conditions than in the field, where the interplay of applied N forms with soil processes and other N sources complicates the picture. Even with the use of urease or nitrification inhibitors, the precise impact of specific fertilizer N forms on agronomic traits remains difficult to predict. Tiller number, a crucial yield component influencing grain-to-straw ratio and grain yield, responds significantly to N fertilization and N form. While tiller abortion often reduces tiller number during the generative growth phase, N dose and form influence tiller formation. Combined ammonium nitrate has shown to be more effective than either N form alone. Phytohormone signaling plays a crucial role: N supply inhibits strigolactone (suppressing tillering) and promotes cytokinin biosynthesis and translocation (promoting tillering), with nitrate having a stronger effect. However, the impact of ammonium or urea on cytokinin translocation and tillering in cereals has been contradictory. This study aims to test the hypothesis that tiller formation in cereal crops can be modulated by the supply of different N forms, under both hydroponic and field conditions. This will involve studying barley's response to nitrate and urea (with a urease inhibitor) in hydroponics, along with cytokinin analysis of xylem sap and translocation studies using artificial cytokinins. The findings will then be translated to field trials with winter wheat to assess their applicability under varying environmental conditions and across different cultivars.

Numerous studies have investigated the impact of different nitrogen forms on plant growth and development. These studies have generally shown that ammonium and nitrate have distinct effects on plant physiology. For example, ammonium nutrition can lead to decreased photosynthesis, respiration, and water use efficiency, while also increasing tolerance to abiotic stresses (Ding et al., 2016; Guo et al., 2007). In contrast, nitrate nutrition tends to stimulate vegetative growth and delay senescence (Escobar et al., 2006). However, the effects of different nitrogen forms are often more pronounced under controlled conditions (e.g., hydroponics) than in the field, where other nitrogen sources and soil processes influence plant nutrition. The use of urease and nitrification inhibitors can mitigate some of the issues associated with urea and ammonium fertilizers, such as ammonia volatilization and nitrous oxide emissions (Zaman et al., 2008; Sigurdarson et al., 2018). Previous research has also demonstrated the importance of phytohormones in regulating tiller formation in cereals. Strigolactones, synthesized under N deficiency, suppress shoot branching and tiller formation. Conversely, cytokinins, synthesized in roots, promote shoot branching and tillering (Le et al., 2020; Ruyter-Spira et al., 2013; Ferreira & Kieber, 2005). Studies have shown that nitrate supply enhances cytokinin translocation from roots to shoots (Rahayu et al., 2005; Walch-Liu et al., 2000), suggesting a potential link between nitrate nutrition and tiller formation via cytokinin signaling. However, the effect of different nitrogen forms on cytokinin translocation and tiller formation in cereals remains somewhat contradictory (Wang & Below, 1996; Chen et al., 1998). This study seeks to address these contradictions and to clarify the mechanistic link between nitrogen form, cytokinin signaling, and tiller formation in cereal crops.

The study employed a two-pronged approach, combining controlled hydroponic experiments with field trials. **Hydroponic Experiments:** Summer barley (cv. Henni) seeds were germinated and transferred to pH-buffered nutrient solutions containing different ratios of nitrate and urea (with the urease inhibitor PPD). Plant growth parameters (dry weight, tiller number, individual tiller weight) were measured. Xylem sap was collected to analyze endogenous cytokinin levels using radioimmunoassay (RIA). The impact of external cytokinin application (BA and BAR) on tillering was also assessed under different N treatments. Short-term experiments determined the effect of different N forms on the root-to-shoot translocation of BAR by ELISA. Ammonium and urea concentrations in plant tissues were measured using HPLC and colorimetric methods, respectively. **Field Trials:** Field experiments used winter wheat (cv. Cubus at Langenstein, cv. Schamane at Dörrhof) at two sites in Germany over two years. Starter N dressing was applied as fully stabilized urea (with urease and nitrification inhibitors), urea plus nitrification inhibitor (ammonium-dominated), or ammonium nitrate (nitrate-dominated). Tiller density was determined at the end of vegetative development. Grain yield and other yield components were measured at harvest. A separate trial screened 22 elite winter wheat cultivars to examine genotypic variation in tillering response to different N forms. Climate data was recorded to assess the influence of environmental conditions on tillering. **Statistical Analysis:** Statistical significance was assessed using appropriate statistical methods (Tukey's test) considering variations in dry weight, tiller numbers, cytokinin levels, and potassium levels.

Hydroponic experiments with barley demonstrated a clear relationship between nitrate supply, cytokinin translocation, and tiller number. Exclusive nitrate supply resulted in significantly higher tiller numbers and shoot biomass compared to urea or ammonium treatments. This difference correlated closely with the root-to-shoot translocation rates of active cytokinin forms (zeatin and zeatin riboside), while less active forms (isopentenyl adenine and isopentenyl adenosine) were less affected by N form. The addition of the synthetic cytokinin analog BA enhanced tillering only in nitrate-fed plants, suggesting that urea may inhibit either cytokinin uptake or translocation. Short-term experiments with BAR confirmed that nitrate significantly enhanced the translocation of this cytokinin analog from roots to shoots. Field trials with winter wheat showed a dose-dependent increase in tiller number with nitrate fertilization, substantially exceeding tiller numbers under urea-based fertilization in most scenarios. The superiority of nitrate fertilization varied across environments, with environmental factors and cultivar impacting tiller number. There was substantial genotypic variation in the response of winter wheat cultivars to different N forms, indicating potential for exploiting this trait in crop breeding. While fully stabilized urea resulted in higher grain yield than ammonium nitrate in some conditions, this is likely attributable to conditions favoring survival and grain development over additional tiller development. In other conditions, the lower tiller densities generated by urea restricted yield potential.

This study provides strong evidence that the choice of N fertilizer form influences tiller number in cereal crops, primarily through its effects on cytokinin signaling. The close correlation between nitrate supply, enhanced cytokinin translocation, and increased tillering in both hydroponic and field experiments supports this conclusion. The observation that external cytokinin application only stimulated tillering in nitrate-fed barley suggests that the effect of nitrate is not solely on cytokinin synthesis, but also on its translocation. The increased translocation of the cytokinin analog BAR under nitrate conditions further points towards a role of nitrate in regulating cytokinin xylem loading. The differential effects of N forms on tiller number are likely due to their influence on specific regulatory steps in cytokinin biosynthesis and translocation. The contrasting responses of different wheat cultivars to the N form highlights the importance of genotypic factors in the N form-dependent control of tillering. The varying influence of weather conditions on tiller number and grain yield emphasizes the need for site-specific fertilization strategies, taking into account the prevailing environmental conditions and stage of plant development. The higher grain yields seen in urea-fertilized plots in certain conditions appear to be related to the effects of the tiller density on drought tolerance and water use efficiency, particularly during the grain filling stage.

This research demonstrates that manipulating the nitrogen form in fertilizers can effectively modulate tiller number in cereal crops. Nitrate-based fertilization generally promotes tillering via enhanced cytokinin translocation, whereas urea tends to suppress tillering. This effect is influenced by both environmental conditions and cultivar genotype. The findings suggest that the choice of N fertilizer form can serve as a valuable tool for managing tiller number, a key yield component, particularly in the context of a changing climate with increased weather variability. Future research should focus on identifying the molecular mechanisms underlying the N-form dependent regulation of cytokinin translocation and on incorporating this knowledge into crop breeding programs to improve yield stability under varying environmental conditions. Investigating the optimal N-form strategies under different environmental scenarios will also be crucial for enhancing agronomic efficiency.

The study primarily focused on two cereal species (barley and wheat) under specific environmental conditions. The generalizability of the findings to other cereal species and a broader range of environmental conditions requires further investigation. While urease inhibitors were used to mitigate urea degradation, microbial processes in the soil could still have influenced the availability of different N forms in the field trials. The study examined tiller number as a primary yield component, but other yield parameters such as grain weight and grain number also affect overall yield. Further research considering additional yield components is necessary for a complete understanding of the implications of different N forms on crop yield.