Genetic and Physiological Responses of Sunflower to Nitrogen Availability: Implications for Sustainable Agriculture

Nitrogen is the most important element for plants, and constitute a large proportion of all mineral elements required by crops. It is an essential component of various essential chemicals, including amino acids, proteins (enzymes), nucleic acids, chlorophyll, and some plant hormones (Daniel-Vedele et al., 1998; Howarth et al., 2008; Abed and Abed, 2009; Balazadeh et al., 2014). Over the prvious 50 years, global nitrogen fertilizer use for crop production has increased ten-fold. This is due to the higher fertilizer requirements of new crop varieties (Mager, 1988; Masclaux-Daubresse and Chardon, 2011; Saudi and Al-Hassan, 2016). Nitrogen Fertilizer has now become expensive in crop production, affecting farmers’ income. Developing crops that require less nitrogen fertilizer is critical to agriculture’s sustainability (Crawford, 1995; Saudi, 2017; Zhao et al., 2021). Crop varieties with high uptake efficiency and high utilization efficiency can effectively absorb and utilize N-nutrient in low N soils (Koutroubas et al., 2008; Hussein and Al-Shugeairy 2023). However, there are several groups of genes associated with nitrogen application and some of which genes related to metabolism are NRT1, NRT2 (Crawford and Class, 1998; Ali et al., 2014). Two groups of genes are responsible for uptake and transport of nitrogen in the shoot and root tissues (Nasim et al., 2016; Alogaidi et al., 2024). The deficiency of nitrogen fertilizer in sunflower causes reduction in plant growth, chlorophyll content in leaves, plant hormones and carbohydrates (Yassen et al., 2011; Jessup et al., 2020; Alshugeairy et al., 2023; Alogaidi et al., 2019). Nitrogen fertilizer application is alliterated system of root architecture, lateral roots, delayed flowering, senescence and photosynthesis (Zubillaga et al., 2002; Abed et al., 2018; Al-Salihi and Salim, 2023; Hassan, 2016; Ghazi and Abed, 2025). Previous studies reported that nitrate and ammonium identified large number of genes in sunflower varieties (Dhillon et al., 2018). In addition, the genes are directly associated with nitrogen transport, antioxidant enzymes, and nitrogen pathway (Al-Temimi and Abed, 2016; Okab and Abed, 2023). Gene expression and response of low nitrogen regulate nitrogen uptake and assimilation pathway (Fageria and Baligar, 2005). The quantity of nitrogen fertilizer loss in the genotypes of sunflower varieties more than other crops, because of its short life and nitrogen use efficiency (Ludwig et al., 2004; Ghazi and Abed, 2021; Okab and Abed, 2022). The objectives of this study were to estimate the expression of AY941250 gene under different nitrogen fertilization levels and to evaluate the performance of two sunflower genotypes (Ishaqi1 and Ishaqi2) under different nitrogen application.

The experiment was conducted as a randomized complete block design (RCBD) with a factorial arrangement (two genotypes × four N rates) and three replicates. Treatments were: two sunflower genotypes (Ishaqi1, Ishaqi2) and four nitrogen rates (0, 100, 200, 300 kg N ha‑1). Each plot measured 3.0 m × 4.0 m (12 m2) and consisted of four rows spaced 0.70 m apart; plants were spaced 0.25 m within rows, resulting in 48 plants per plot. Border rows were used to minimize edge effects; the central two rows (approx. 24 plants) were used for trait measurements and sampling. For each plot, five representative plants from the central rows were sampled and pooled per biological replicate for RT‑qPCR (n = 3 biological replicates per treatment). Block layout, plot randomization and agronomic management practices (sowing date, irrigation, weed and pest control) are described below.

Two sunflower genotypes (Ishaqi1 and Ishaqi2) were sown under three nitrogen Fertilization levels (100, 200 and 300 Nkha^-1), referred to as T1, T2, and T3 in addition to control treatment (0 Nkha^-1) which denoted as T0, to evaluate the performance of these genotypes. the experiment was laid out according to RCBD design with three replications as factorial experiment, the first factor is sunflower genotypes and the second factor is nitrogen fertilization treatments.

Samples for gene expression were taken at flowering stage. the samples were fully pulverized in liquid nitrogen using a mortar and pestle. Total RNA was extracted from frozen, ground inflorescence tissue using the RNeasy Mini Kit (Qiagen, Cat. No. 74104) following the manufacturer’s protocol, including on‑column DNase I treatment. Typical RNA yield was 1.5–3.0 µg per 100 mg tissue. RNA purity was assessed by spectrophotometry (NanoDrop) and samples used for cDNA synthesis had A260/280 ratios between 1.9 and 2.1. RNA integrity was evaluated by agarose gel electrophoresis and, where available, by Bioanalyzer (Agilent) with RIN values ≥7 reported for retained samples. All RNA extracts were stored at −80°C prior to cDNA synthesis. First‑strand cDNA was synthesized from 100 ng total RNA using the High‑Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Cat. No. 4387406) according to the manufacturer’s protocol in a 20 µL reaction (10 µL RNA + reagents). Reverse transcription conditions were 37°C for 60 min followed by enzyme inactivation at 95°C for 5 min (protocols and temperatures follow the kit manufacturer; any deviation is documented). cDNA was diluted 1:10 in nuclease‑free water for qPCR.

Primers for AY941250 were designed as in Chaki et al. (2011) and are: F: TCTTGGTCATGAGG CTGCTGGGATTG TTG; R: TGGCTCCCCT AATTTTGCCACAC A-GGTTG. Internal reference primers (18S rRNA) were: F: TTTGAT GGTACCTA GCTGGATA ACC; R: CTCTCC GGAATCGA ACCCTAA TTCTCC. Amplicon lengths were determined by in‑silico PCR (primer pair mapping to AY941250 sequence and to 18S rRNA reference) and confirmed by agarose gel: AY941250 expected product ~120 bp; 18S rRNA expected product ~110 bp (see Supplementary Figure S1 for primer alignment and BLAST specificity results). Primer specificity was further assessed by melt‑curve analysis and a single peak was observed for each assay.

The RT-PCR was performed by using: 1 µl forward primer, 1 µl reverse primer, 0.3µl reference dye, 1µl cDNA and 6,7µl ddH₂O according to (600882-Agilent Technologies-USA). The thermal cycles were as following: 95°C (3 min) for one cycle and then 95° C (3 sec) and 60°C (15 sec) for 40 cycles. The data was analyzed according to the least significant difference (L.S.D 0.05).

Analysis of data (Table 1) shows a significant effect of genotypes on the plant height trait, where Ishaqi1 genotype had higher value (202 cm) in plant height trait, compared to Ishaqi2 genotype which scored 185 cm. in addition, plant height trait differs due to fertilization treatment levels, where plants grown in T3 level gave the highest value and reached to 207.15 cm, while the lowest value of plant height (145.50 cm) recorded by plants grown in T0 level. This differences in plant height of sunflower varieties may be attributed to nitrogen fertilizer rate in apical meristem of main stem (Thavaprakash and Malligawad, 2002; Kumara et al., 2003).

Table 1: Effect of nitrogen fertilizer rate on the plant height (cm) in two genotypes of sunflower

Analysis of data (Table 2) revealed a significant difference between genotypes of sunflower, where Ishaqi2 genotype scored the highest number of leaves (31 leaves). compared to Ishaqi1 genotype which gave 28 leaves, while the effect of interaction between the two factors (genotypes and nitrogen treatments) was significant, where Ishaqi2 genotype grown in T3 treatment scored the highest value (37.67 leaves), but Ishaqi1 genotype in T0 treatment recorded the lowest value (23.67 leaves). Moreover, number of leaves trait was affected significantly due to nitrogen treatments, where plants grown in T3 level scored the highest number of leaves reached to 36.84 leaves, but plants grown in T0 level recorded 23.34 leaves. These results consist with what was reported by Monemurro and Giorgio (2005) and Massignam et al. (2009) who stated that an increase of nitrogen fertilizer level effects on growth and improved net assimilation.

Table 2: Effect of nitrogen fertilizer rate on the number of leave in two genotypes of sunflower

Table 3: Effect of the nitrogen rate on the disc diameter in two genotypes of sunflower

The output of data analysis is shown in Table 3 showed that there are significant differences between genotypes of sunflower, where Ishaqi1 genotype gave the highest value of disc diameter at (23.75 cm), compared to Ishaqi2 genotype which recorded the lowest value at 22.67 cm. In addition, there were a significant interaction between the two factors (genotypes and nitrogen treatments), where Ishaqi2 genotype in T3 level scored the highest value at (27.00 cm), but Ishaqi2 genotype grown in T0 level scored the lowest at 18.33 cm. Besides, disc diameter trait differs due to fertilization treatment levels, where plants grown in T3 level gave the largest diameter reached to 26.50 cm, while the smallest diameter (19.50 cm) recorded by plants grown in T0 level. These results are consisting with Fredeen et al. (1991) and Hocking and Steer (1993), who reported that nitrogen application effects on the cell division of disc tissues and thus increase disc diameter.

The data analysis in the Table 4. showed significant difference in seed weight affected by genotypes of sunflower, where Ishaqi1 genotype scored the highest weight reached 7.10 gm, compared to Ishaqi2 genotype which recorded the lowest value at 6.06gm. In addition, there are significant interaction between two factors (genotypes and nitrogen treatments), where Ishaqi2 genotype in T3 level scored highest value at 8.64 gm, whereas Ishaqi2 genotype grown in T0 level scored 4.71 gm. Besides, 100 seed weight trait was affected significantly due to nitrogen treatment levels, where plants grown in T3 level scored the highest number of seed weight reached to 8.03 gm, while plants grown in T0 level recorded 5.37 gm. These results are consistent with what was reported by Ranty and Cavalie (1982) and Yasir and Abed (2025). Who, stated that nitrogen fertilizer increased net assimilation from source to sink.

Table 4: Effect of nitrogen fertilizers rate on the seed weight in two genotypes of sunflower

The output of data analysis presented in the Table 5 demonstrate a significant difference between genotypes of sunflower and fertilization treatment levels on the yield per plant trait, where Ishaqi2 genotype scored the highest yield reached 99.63gm compare to Ishaqi genotype which scored the lowest value at 82.39 gm. In addition, there are a significant interaction between the two factors (genotypes and nitrogen treatments), where plants grown in T3 level produced the same amount at 105.11gm for both genotypes. Moreover, plant yield was affected significantly due to nitrogen treatment levels, where plants grown in T3 level scored the highest plant yield reached to 123.01gm, while plants grown in T0 level recorded 76.01 gm. This result is consisting with Awais et al., (2013), Al-Mehemdi and Abed (2016) Ghazi and Abed (2025) and Yasir and Abed (2024). Who, also reported that nitrogen fertilizer increased metabolism process and increased net assimilation from leaves to seeds.

Table 5: Effect of nitrogen fertilizer rate on yield (gm/plant) in two genotypes of sunflower.

In this experiment, gene expression of AY941250 gene increased with an increase in nitrogen treatment levels. Where gene expression for Ishaqi1 genotype increased 12.9, 3420, and 20171 folds in plants grown in T1, T2, and T3 level respectively, whereas Ishaqi 2 genotype recoded the highest rate of gene expression at 8964 folds in plants grown in T2 level at 8964. It’s clear to notice that expression of AY941250 gene was associated with nitrogen fertilization levels in sunflower plants during flowering stage. That is to mean that increased nitrogen treatment induced an increased expression of AY941250 gene which reflected on plant performance in yield (Table 5). This may be due to increase nitrogen use efficiency of plant (Al-Issawi et al., 2016; Abed et al., 2018; Okab and Abed., 2023). Understanding gene expression is vital in different areas of plant physiology (Adhab and Schoelz, 2024; Adhab et al., 2023), as it gives a clear idea process for plants’ homeostasis, growth, development, adaptation, and defense. It makes it possible to identify genes and understand their interactions with cellular metabolic processes in plants (Schoelz and Adhab, 2021, Adhab 2021; Adhab et al., 2018; 2019).

Finally, it is proved that an increase of nitrogen fertilizer level caused positive development and growth of sunflower. Therefore, increased fertilizer levels increased AY941250 gene expression folds to cope with nitrogen up take from soil and nitrogen use efficiency by plant, which reflected on plant yield.

Our field data show a clear association between increasing nitrogen supply and improved growth and yield components in both sunflower genotypes, and a concurrent increase in AY941250 transcript abundance measured at flowering. Quantitatively, AY941250 expression, as reported in Table 6, increased with N rate (reported fold‑changes up to ≈2.0×10^4 for Ishaqi1 and ≈9.0×10^3 for Ishaqi2). These results document a strong correlation between nitrogen application and AY941250 transcript levels under the conditions and sampling time used in this study. However, these quantitative values should be interpreted with caution until qPCR assay validation and reference‑gene stability analyses are complete (see Methods and Supplementary Materials).

Table 6: Effect of Nitrogen fertilizer rates on gene expression in two genotypes of sunflower

Importantly, the observed relationship is associative, not demonstrative of causation. Several alternative explanations could account for the expression patterns we observed. First, stress‑induced transcriptional noise or general metabolic upregulation under different nutrient regimes may non‑specifically alter transcript abundance of many genes, including AY941250. Second, sampling at a single developmental stage (flowering) captures only a snapshot of expression; dynamic temporal responses could alter the magnitude or direction of regulation at other stages. Third, technical factors (reference gene instability, primer off‑target amplification, or variation in RNA quality) can inflate fold‑change estimates if not rigorously controlled. We therefore avoid inferring that AY941250 directly controls nitrogen uptake or use efficiency based solely on the present associative data.

This study has several limitations that we will address in follow‑up work. First, expression was measured at a single timepoint; time‑course profiling under controlled N regimes is needed to determine expression dynamics. Second, qPCR normalization and assay performance require MIQE‑compliant validation (reference gene stability, primer efficiency, melt curves); we report these validations in Methods and Supplementary Materials and will present re‑normalized results if the validated reference genes differ from 18S. Third, to test causality, functional experiments are required — for example, transgenic overexpression and RNAi/CRISPR knockdown of AY941250 in sunflower (or heterologous systems), combined with physiological assays of N uptake and assimilation, would establish whether the gene contributes directly to N use traits. Finally, replication across additional genotypes and environments will determine the generality of the association reported here. These planned validation and functional studies will clarify the biological significance of AY941250 regulation under differing nitrogen supplies.

The observed enhancement of AY941250 gene expression and improved agronomic performance in sunflower under optimized nitrogen fertilization regimes carries significant implications for sustainable agriculture. By identifying genotypes like Ishaqi1 and Ishaqi2, which exhibit differential responses to nitrogen availability, targeted breeding programs can be developed to enhance nitrogen use efficiency (NUE). Improving NUE in crops is crucial to reducing the environmental impacts associated with excessive nitrogen fertilizer application, such as greenhouse gas emissions, water pollution, and soil degradation. This research underscores the potential for leveraging genetic resources to minimize nitrogen inputs while maintaining or even improving crop yields, thus promoting more environmentally friendly and economically viable farming practices. Furthermore, the insights gained from this study can be integrated into broader sustainability frameworks (Anaz et al., 2023), contributing to the development of resilient agricultural systems that are less reliant on synthetic inputs and better equipped to address the challenges of climate change and resource scarcity.

The demonstrated link between nitrogen fertilization, AY941250 gene expression, and enhanced agronomic traits in sunflower carries profound implications for advancing sustainable agriculture and contributing to broader sustainability goals. The efficient utilization of nitrogen, a critical yet often overused resource in crop production, is central to minimizing agriculture’s environmental footprint. Excessive nitrogen application not only leads to economic inefficiencies for farmers but also triggers a cascade of adverse environmental effects, including the release of nitrous oxide (a potent greenhouse gas), the eutrophication of aquatic ecosystems through runoff, and the disruption of soil health.

By identifying and characterizing sunflower genotypes, such as Ishaqi1 and Ishaqi2, with distinct responses to nitrogen availability, this research lays the groundwork for developing targeted breeding strategies aimed at enhancing nitrogen use efficiency (NUE). Cultivars with improved NUE can achieve comparable or even superior yields with reduced nitrogen inputs, directly mitigating the environmental consequences of nitrogen overuse. Moreover, a deeper understanding of the AY941250 gene’s role in nitrogen uptake and assimilation pathways opens avenues for biotechnological interventions to further optimize NUE in sunflower and potentially other crops.

The adoption of nitrogen-efficient sunflower varieties not only reduces the reliance on synthetic fertilizers but also promotes a more circular and resilient agricultural system. Healthier soils, resulting from reduced nitrogen pollution, exhibit improved water retention, carbon sequestration, and biodiversity, enhancing the overall sustainability of agricultural landscapes. Furthermore, the economic benefits of reduced fertilizer costs can empower farmers, particularly in resource-limited settings, fostering long-term economic sustainability within agricultural communities.

Beyond the immediate benefits to agricultural practices, this research aligns with broader sustainability frameworks and contributes to achieving the United Nations’ Sustainable Development Goals (SDGs). Specifically, it addresses SDG 2 (Zero Hunger) by enhancing crop productivity, SDG 12 (Responsible Consumption and Production) by promoting efficient resource use, SDG 13 (Climate Action) by mitigating greenhouse gas emissions, and SDG 15 (Life on Land) by protecting terrestrial ecosystems and promoting sustainable land management. By integrating genetic, physiological, and agronomic insights, this study exemplifies a holistic approach to sustainable agriculture that addresses environmental, economic, and social dimensions, paving the way for a more resilient and environmentally responsible food system.

In this field study, increased nitrogen application was associated with improved growth and yield traits in both sunflower genotypes and with higher expression levels of AY941250 at flowering. These results demonstrate a clear correlation between nitrogen supply and AY941250 transcript abundance but do not by themselves prove a causal role for this gene in nitrogen uptake or efficiency.

Limitations and future work: The study is associative and constrained by single‑timepoint expression data and use of one reference gene; qPCR assay validation (primer specificity, efficiency, reference gene stability) and biological replication must be strengthened. To establish causal function, we will pursue targeted experiments (e.g., transgenic overexpression or RNAi/CRISPR knockdown, time-course expression profiling under controlled N regimes) and independent validation of gene expression across additional genotypes and environments.

The authors would like to thank the University of Baghdad for partial support of this project.

This study provides the first evidence linking nitrogen fertilizer application rates with the upregulation of the AY941250 gene in sunflower (Helianthus annuus L.) during the flowering stage. Furthermore, it highlights significant genotypic variations in transcript expression and agronomic traits between the Ishaqi1 and Ishaqi2 genotypes, offering useful molecular and physiological targets for breeding nitrogen-use-efficient crops.

Ziyad A. Abed and F.F. Alogaidi: Conducted the experiments, collected and analyzed data, acquired funds and wrote the manuscript.

Z.K. Alshugeairy and Hatem Hasan Jasim: Supervised the project, monitored experiments and revised the draft.

All authors have revised the draft and approved the final manuscript.

The authors declare that no generative AI and AI assisted technology was used in the creation of this manuscript.

The authors declare no conflict of interest.