Effects of nitrogen doses on stomatal characteristics, chlorophyll content, and agronomic traits in wheat (Triticum aestivum L.)

Introduction

Cereals and products obtained from seeds are an indispensable source of basic nutrition for the majority in Turkey, as in many countries of the world. Cereals are the most important nutritional sources in human nutrition. More than 50% of the calorie input in nutrition is covered by seeds. Wheat is an extremely useful plant in terms of meeting people’s calorie and protein needs. At 20% of the world’s calorie and protein consumptions is met by the wheat plant. Wheat (Triticum L.) is one of the first cultivated cereal and has been the main food of people in Europe, Western Asia and North Africa for thousands of years. In Turkey, wheat ranks first in terms of both cultivation area and production potential. According to 2020 data, wheat constituted 28% of the seed cultivation area and 65% of seed production while wheat is cultivated in 44% of the total cultivated area in Turkey (FAOSTAT, 2020).

The seed demand increases by the population. Both increasing yields for the rising demand for seed production and ensuring sustainability are the biggest challenges in agriculture. Fertilization is required for high efficiency in agricultural systems.

Nitrogen (N) is the most important macronutrient limiting plant growth (Hawkesford, 2014; Yang et al., 2019). N greatly boosts crops (Lachutta & Jankowski, 2024). N fertilization delays leaf aging by reducing N release from the leaf after flowering and provides carbohydrates for seed filling (Li et al., 2012). Plant growth and photosynthesis are inhibited in N deficiency, which causes a decrease in yield (Jin JiYun & He Ping, 1999; Shangguan, Shao & Dyckmans, 2000). Also, vegetative and reproduction are delayed, and yield components such as yield and agronomic traits, the number of spikes per plant, number of seeds per spike, single seed weight and number of seeds per plant decrease (Wang et al., 2021). Additionally, N deficiency reduces leaf area index and protein content of the plant and seed (Steer & Harrigan, 1986). N deficiency increases flower abscission, reduces seed formation and inhibits seed growth (Dordas & Sioulas, 2008; Ciampitti & Vyn, 2011). N accumulation and productivity increase are closely related to each other (Lemaire et al., 2021). The fact that N uptake by plants affects the increase in productivity shows (Kayan et al., 2020) that N physiologically plays a role in the formation of new plants (Sinclair, Rufty & Lewis, 2019).

Crop production occurs through photosynthesis and dry matter accumulation depending on leaf area index (Lee & Tollenaar, 2007; Sadras et al., 2016; Thomas & Ougham, 2014). Photosynthetic products constitute the main source of plant biomass. Therefore, as photosynthesis in the leaf increases, so does the biomass obtained (Noor et al., 2023). N affects leaf area index development (Steer & Harrigan, 1986). Seed N concentration and seed yield are the main targets in wheat breeding, but it has been difficult to increase them simultaneously due to the negative genetic relationship between them (Mu et al., 2018). When N is applied at optimum levels in wheat, 35–42% of the N accumulated in the soil is in the leaf lamina, 14–20% in the leaf sheath, 20–31% in the stem and 16–23% in the spike (Slafer, Andrade & Satorre, 1990; Bogard et al., 2010; Pask et al., 2012).

Stomata are natural microscopic pores found in the leaf that serve to connect near part of the leaf to the external environment (Barraclough et al., 2010; Gaju et al., 2014). Stomata are surrounded by a pair of protective cells and enable gas exchange between the inner part of the leaf and the ex-ternal environment. The photosynthetic capacity of plants depends on stomatal size and density. The stomatal density of the leaf, that is, the number of stomata per unit area of the leaf, is an important parameter affecting gas exchange and is important in terms of the water and carbon relations of the plant (Hedrich & Shabala, 2018; Lawson & Vialetchabrand, 2019). Stomatal movement is closely related to N. Nitrate is the main source in plant development, is important in regulating stomatal movements (Rogiers, Hardie & Smith, 2011). It has been reported by many researchers that high N fertilisation increases photosynthesis and plant growth (Evans, 1989; Doheny-Adams et al., 2012; Mu & Chen, 2021). At 75% of the N in the leaf is found in chloroplasts and is a part of ribulose bisphosphfate carboxylase (Rubisco). Reduced photosynthesis in the presence of low N is generally due to a decrease in chlorophyll content and Rubisco activity (Huber, Sugiyama & Alberte, 1989; Cechin & De Fátima Fumis, 2004). Since N is a component of chlorophyll (Robinson & Burkey, 1997), in some species wheat (Triticum aestivum L.), stomatal conductance increases with N (Evans & Terashima, 1987).

This study is based on the hypothesis that increasing N doses in wheat influences stomatal structure and chlorophyll content.

Materials and Methods

Experimental design and materials

The field study was conducted at the experimental farm of Ordu University, Faculty of Agriculture, Turkey (40°58′09″ N 37°56′19″ E) in two wheat growing seasons (2020–2021 and 2021–2022). The experiment was carried out according to the factorial randomized complete block design with 3 repetitions and for 2 years. The soil was neutral in response to (pH 5.78–6.62), somewhat salty (0.034–0.046%), with very low N (0.012–0.016 kg ha⁻¹), low phosphorus (0.20–0.19 kg ha⁻¹), high potassium (4.07–4.28 kg ha⁻¹), and saturation (%) at 2020–2021 and 2021–2022 growing periods, respectively (Table 1). Nine wheat varieties (Alpu-2001, Soyer-02, Kate-A1, Bezostaja-1, Altay-2000, Müfitbey, Nacibey, Harmankaya-99, Sönmez-2001) were used in the study, which were grown varieties in Turkey. In the experiment, 60 kg phosphorus and 50 kg calcium fertilizers were applied to the hectare before sowing and half of them were applied with sowing and the other half before emergence. Three rates of N dose (0 (control), 50 and 100 kg ha⁻¹) were applied as granules of urea (34.5% N). The experimental plots were 3 m × 5 m and consisted of 5 rows 0.5 m apart. Border rows were not included for any sampling. Potassium, as potassium sulfate (50% potassium) and phosphorus, as calcium superphosphate (43% phosphorus), were applied for each plot as basal fertilizer at rates.

Table 1:

Some chemical characteristics of the soil in the field area.

Years	Salt (%)	*Saturation (%)	pH	N (kg ha−1)	P (kg ha−1)	K (kg ha−1)
2021	0.034	118.8	5.78	0.012	0.20	4.07
2022	0.046	116.6	6.62	0.016	0.19	4.28

DOI: 10.7717/peerj.18792/table-1

Note:

* Saturation > 110% is highly-clay.

The meteorological data during the growing seasons of wheat were given in Fig. 1. The region of investigation has a temperate climate, humid summers followed by rainy and warm winters. During the 2020–2021 growing season, the mean humidity ranged between 65.3% and 78.2%, while as in the 2021–2022 growing season, it ranged between 60.9% and 77.1%. Average precipitation and temperature values obtained in all years were close to each other (Fig. 1).

Methods of determination

Statistical analysis

In order to decide on the analysis method to be performed, Skewness and Kurtosis values were examined (±3.29) and it was determined that the data were compatible with normal distribution (±1.36). Factorial randomized complete block design (RCBD) (Model 1) was used in the study and variety and N dose were considered as fixed factors. Comparisons between years were determined by t test.

(1) $${\gamma }_{ijk}=\mu +{\alpha }_i+{\beta }_j+{\left(\alpha \beta \right)}_{ij}+{ϵ}_{ijk}$$

$\mathit{\alpha }$: Variety

$\mathit{\beta }$: N doses

$\mathit{\alpha }\mathit{\beta }$: Variety X N dose

$\mathit{ϵ}$: Error

In order to determine the appropriate multiple comparison test, the Levene homogeneity test was performed and when it was determined that the data were homogeneous, the TUKEY multiple comparison analysis was decided. The TUKEY (Model 2) multiple comparison test was used at the 0.01 or 0.05 level, depending on the degree of significance, if there was a significant difference between the components.

(2) $$HSD=qx\sqrt{{\displaystyle \frac{MSW}{n}}}$$

Analysis of variance was performed with SPSS 24.0 software (SPSS Inc., IL, USA).

In this study, principal component analysis (PCA) was applied to enable better analysis of the high-dimensional dataset and to optimize the impact of dimensions during the modelling process. PCA evaluated the relationships among the variables in the dataset and identified a few principal components that explain the variance best.

PCA was conducted using the “FactoMineR” package (Lê, Josse & Husson, 2008) and visualized with “factoextra” (Kassambara & Mundt, 2020) in R. The dataset was standardized to ensure comparability across variables. The N dose groups (N0: 0 kg ha⁻¹, N1: 50 kg ha⁻¹, N2: 100 kg ha⁻¹) and wheat varieties (V1–V9) were included as a grouping factor separately, and confidence ellipses were added to illustrate variability within each group.

Variable contributions to the first two dimensions (Dim1 and Dim2) were analyzed to identify key traits driving the observed patterns. Results were visualized with a biplot combining individuals and variables, and a variable contribution plot was created to highlight the relative influence of each variable.

Results

All parameters except plant height and spike length values were not statistically affected by the years and no significant difference was observed between 2020–2021 growing season and 2021–2022 growing season (Figs. 2A, 2B).

Seed yield and agronomic traits

The effects of variety and N dose on yield and agronomic traits of wheat plant are given in Tables 2 and 3 both years. With the increase in nitrogen dose, an increase was observed in all parameters examined compared to the control and N2 dose gave the highest values. When the N dose increased from 0 to 100 kg ha⁻¹, the change in seed yield increased from 3,659 to 5,511 kg ha⁻¹ (increased 50.61%) at first year (Table 2), while from 3,859 to 5,696 kg ha⁻¹ rise was obtained (increased 47.60%) at second year (Table 3). The highest seed yields were obtained from Müfitbey (5,044 kg ha⁻¹ for 2020–2021 and 5,222 kg ha⁻¹ for 2021−2022 growing seasons respectively) and Kate-A1 (4,900 kg ha⁻¹ for 2020–2021 and 5,067 kg ha⁻¹ for 2021–2022 growing seasons, respectively) varieties (Tables 2 and 3).

Table 2:

Effects of treatment on yield and yield components of wheat in 2020–2021 (1^st year) growing season (N_n mean ± sd).

Treatment	Seed yield (kg ha−1)	Plant height (cm)	1,000 seed weight (g)	Number of spikelets (number/spike)	Number of grains (number/spike)	Spike length (cm)	Number of spikes (number/m2)
N0	3,659 ± 327c	72.25 ± 7.93c	39.18 ± 2.67c	12.63 ± 1.60c	32.78 ± 4.12c	7.29 ± 0.92c	268.11 ± 38.58c
N1	4,689 ± 392b	85.24 ± 10.34b	41.27 ± 3.00b	15.63 ± 2.34b	41.85 ± 1.75b	8.44 ± 1.00b	313.15 ± 47.88b
N2	5,511 ± 409a	96.94 ± 11.03a	42.81 ± 3.21a	19.48 ± 2.34a	51.07 ± 5.10a	9.35 ± 1.19a	380.63 ± 35.83a
Mean	4,619 ± 848	84.81 ± 14.05	41.08 ± 3.29	15.91 ± 3.51	41.90 ± 8.45	8.36 ± 3.13	320.63 ± 61.73
V1	4,289 ± 775c	81.25 ± 7.31bc	41.09 ± 2.10cd	17.11 ± 2.57a	42.78 ± 8.56	8.22 ± 0.92b	316.00 ± 66.62ab
V2	4,644 ± 1,041abc	78.53 ± 12.76c	37.36 ± 0.91f	12.56 ± 1.67c	42.22 ± 8.97	7.68 ± 1.06b	313.67 ± 53.68ab
V3	4,900 ± 1,005ab	78.63 ± 11.05c	36.83 ± 1.34f	15.67 ± 2.50ab	42.33 ± 10.15	8.13 ± 1.33b	310.56 ± 40.68ab
V4	4,444 ± 979bc	86.20 ± 10.40abc	42.63 ± 2.89b	16.56 ± 3.64a	39.67 ± 8.63	10.20 ± 1.14a	322.00 ± 70.25ab
V5	4,478 ± 689bc	93.26 ± 14.28ab	39.43 ± 1.11e	14.00 ± 3.87bc	42.67 ± 7.26	8.30 ± 1.05b	353.56 ± 30.45ab
V6	5,044 ± 914a	76.70 ± 9.73c	42.31 ± 1.01bc	16.22 ± 4.18a	42.22 ± 8.67	8.64 ± 1.67b	357.44 ± 90.63a
V7	4,522 ± 670abc	84.90 ± 11.69abc	40.51 ± 2.32de	17.33 ± 3.54a	41.89 ± 9.64	8.10 ± 1.10b	322.89 ± 54.99a
V8	4,644 ± 639abc	89.20 ± 19.56abc	45.92 ± 2.37a	17.78 ± 2.91a	41.89 ± 8.72	8.40 ± 1.25b	280.44 ± 29.66b
V9	4,611 ± 948abc	94.64 ± 17.99a	43.70 ± 1.41b	16.00 ± 4.00a	41.44 ± 8.86	7.56 ± 0.89b	309.11 ± 79.01ab
Mean	4,619 ± 848	84.81 ± 14.05	41.08 ± 3.29	15.91 ± 3.51	41.90 ± 8.45	8.36 ± 3.13	320.63 ± 61.73
N doses (n = 3)	**	**	**	**	**	**	**
Variety (n = 9)	**	**	**	**	ns	**	**
N doses x Variety (n = 27)	**	ns	**	**	ns	ns	*

DOI: 10.7717/peerj.18792/table-2

Note:

a, b; different letters in the same column indicate the difference between averages. V1, Alpu-2001; V2, Soyer-02; V3, Kate-A1; V4, Bezostaja-1; V5, Altay-2000; V6, Müfitbey; V7, Nacibey; V8, Harmankaya-99; V9, Sönmez-2001; Nitrogen doses, N0, Control; N1, 50 kg ha⁻¹; N2, 100 kg ha⁻¹; *P ≤ 0.05; **P ≤ 0.01; ns; non significant; sd, standard deviation.

Table 3:

Effects of treatment on yield and yield components of wheat in 2021–2022 (2^nd year) growing season (mean ± sd).

Treatment	Seed yield (kg ha−1)	Plant height (cm)	1,000 seed weight (g)	Number of spikelets (number/spike)	Number of grains (number/spike)	Spike length (cm)	Number of spikes (number/m2)
N0	3,859 ± 295c	85.66 ± 8.35b	39.71 ± 2.51c	12.74 ± 1.70c	34.44 ± 4.68c	6.29 ± 0.92c	295.11 ± 38.58c
N1	4,867 ± 394b	86.13 ± 9.93b	42.01 ± 2.88b	15.74 ± 2.18b	43.52 ± 2.86b	7.44 ± 1.00b	340.15 ± 47.88b
N2	5,696 ± 416a	99.27 ± 7.63a	43.33 ± 2.91a	19.59 ± 2.39a	52.63 ± 5.85a	8.35 ± 1.19a	407.63 ± 35.83a
Mean	4,807 ± 840	90.35 ± 10.67	41.68 ± 3.12	16.02 ± 3.51	43.53 ± 8.75	7.36 ± 1.33	347.63 ± 61.73
V1	4,533 ± 675c	93.75 ± 7.34ab	40.98 ± 2.06c	18.11 ± 2.57a	45.78 ± 8.56a	7.22 ± 0.92b	343.00 ± 66.62ab
V2	4,833 ± 1,050abc	93.23 ± 8.44ab	37.85 ± 1.06d	13.56 ± 1.67d	45.22 ± 8.97a	6.68 ± 1.06b	340.67 ± 53.68ab
V3	5,067 ± 1,021ab	97.65 ± 2.72a	38.00 ± 1.36d	14.67 ± 2.50cd	40.33 ± 10.15ab	7.13 ± 1.33b	337.56 ± 40.68ab
V4	4,644 ± 979bc	87.49 ± 9.69ab	43.63 ± 2.89ab	15.56 ± 3.64bcd	37.67 ± 8.63b	9.20 ± 1.14a	349.00 ± 70.25ab
V5	4,667 ± 712bc	93.70 ± 7.90ab	40.32 ± 0.96c	15.00 ± 3.87cd	44.67 ± 7.26a	7.30 ± 1.05b	380.56 ± 30.45a
V6	5,222 ± 922a	81.34 ± 11.21b	43.27 ± 0.85b	17.22 ± 4.18ab	44.22 ± 8.67ab	7.64 ± 1.67b	384.44 ± 90.63a
V7	4,711 ± 653bc	87.52 ± 12.72ab	41.17 ± 2.54c	18.33 ± 3.54a	44.89 ± 9.64a	7.10 ± 1.10b	349.89 ± 54.99ab
V8	4,800 ± 634abc	90.28 ± 11.34ab	44.92 ± 2.28a	16.78 ± 2.91abc	44.89 ± 8.72a	7.40 ± 1.25b	307.44 ± 29.66b
V9	4,789 ± 949abc	88.22 ± 14.98ab	45.01 ± 1.62a	15.00 ± 4.00cd	44.11 ± 8.55ab	6.56 ± 0.89b	336.11 ± 79.01ab
Mean	4,807 ± 840	90.35 ± 10.67	41.68 ± 3.12	16.02 ± 3.51	43.53 ± 8.75	7.36 ± 1.33	347.63 ± 61.73
N doses (n = 3)	**	**	**	**	**	**	**
Variety (n = 9)	**	**	**	**	**	**	**
N doses x Variety (n = 27)	**	ns	*	**	ns	ns	*

DOI: 10.7717/peerj.18792/table-3

Note:

a, b; different letters in the same column indicate the difference between averages. V1, Alpu-2001; V2, Soyer-02; V3, Kate-A1; V4, Bezostaja-1; V5, Altay-2000; V6, Müfitbey; V7, Nacibey; V8, Harmankaya-99; V9, Sönmez-2001; Nitrogen doses, N0, Control; N1, 50 kg ha⁻¹; N2, 100 kg ha⁻¹; *P ≤ 0.05; **P ≤ 0.01; ns; non significant; sd, standard deviation.

The plant height was significantly affected by N dose and varieties, but it wasn’t significantly affected by their interactions. As the N dose increased from 0 to 50 kg ha⁻¹, the plant height increased from 72.25 to 85.24 cm (increased 17.97%). Morever its increase from 50 to 100 kg ha⁻¹ resulted in the the plant height growth from 85.24 to 96.94 cm (increased 28.23%) at first year (Table 2). In the second year, these increase rates were measured as 1.00% and 15.26%, respectively (Table 3). While the lowest plant height mean was obtained from Müfitbey wheat variety (76.70 cm for 2020–2021 and 81.34 cm for 2021–2022 growing seasons, respectively), the highest mean plant height was obtained from Sönmez-2001 wheat variety (94.64 cm) at first year and from Kate-A1 wheat variety (97.61 cm) at second year (Tables 2 and 3). When it comes to variety x nitrogen interaction obtained from the highest grain yield (V3), 1,000 grain weight (V8), the number of spikelet (V7) and the number of spike (V6) from N2 treatments (100 kg ha⁻¹) were obtained at first year (Figs. 3A–3D, respectively). N dose, varieties and variety x N dose interactions had significant effects on the 1,000 seed weight. The 1,000 seed weight increased from 5.36% as the N dose rate increased from 0 to 50 kg ha⁻¹ at first year (Table 3). A similar increase rate (5.79%) was also observed at second year (Table 3). According to variance analysis the effect of N doses, varieties and interaction of N with varieties on the number spikelets were significant (Tables 2 and 3, both years). When the N dose increased from 0 to 100 kg ha⁻¹, the change in number of spikelets increased from 12.63 to 19.48 number spike⁻¹ in first year (Table 2) and increased from 12.74 to 19.59 number spike⁻¹ in the second year. When the varieties were compared individually in terms of the number spikelet, the lowest was obtained from Soyer-02 variety (12.56 number spike⁻¹ for 2020–2021 and 13.56 number spike⁻¹ for 2021–2022 growing seasons, respectively (Tables 2 and 3). While the number of grains per spike was significantly affected only by N dose at first year (Table 2), it was affected by variety and N dose at second year (Table 3). As the N dose rate increased from 0 to 50 kg ha⁻¹, the number of grains per spike increased 27.66% and its increase from 50 to 100 kg ha⁻¹ caused 22.03% growth in the number of grains per spike at first year (Table 2). Similar data was found in the second year was found similarly data (26.36% for 2020–2021and 20.93% for 2021–2022 growing seasons, respectively) (Table 3). The spike length was significantly affected by N dose, varieties and their interaction. Increasing the N dose rate from 0 to 50 kg ha⁻¹ resulted in increased spike length (from 7.29 to 8.44 cm for 2020–2021 and 6.29 to 7.44 cm for 2021–2022 growing seasons, respectively). As the N doses increase from 50 to 100 kg ha⁻¹ the rate of increase in spike length (from 8.44 to 9.35 cm for 2020–2021; 7.44 to 8.35 cm for 2021–2022 growing seasons, respectively) (Fig. 4). Among the varieties in terms of spike length, Bezostaja-1 variety stands out both years (Tables 2 and 3). N doses, varieties and their interactions had significant effects on the number of spikes (Tables 2, 3). The number of spikes increased from 268.11 to 380.63 (increased 41.96%) as a result of the N dose rate change from 0 to 100 kg ha⁻¹ at first year, while this increase rate was 38.12% at second year (Tables 2 and 3). When the varieties were compared in terms of number of spikes, the lowest was obtained from Harmankaya-99 variety and the highest was obtained from Müfitbey variety in both years (Tables 2 and 3).

Flag leaf area

N doses, varieties and their interactions had significant effects on the flag leaf area. Flag leaf area increased with increasing nitrogen doses and the highest was found at N2 treatment. The highest flag leaf area was determined from variety Bezostaja-1, while the lowest flag leaf area index was obtained from variety Soyer-02 in both years (Tables 5 and 6).

Table 5:

Effects of treatment on flag leaf area and stomatal characteristics of wheat in 2020–2021 (1^st year) growing season (N_n mean ± sd).

Nitrogen dose	Flag leaf area (cm2)	Abaxial stomata width (µm)	Abaxial stomata length (µm)	Adaxial stomata width (µm)	Adaxial stomata length (µm)	Epidermal cell (no./mm2)	Stomata density (no./mm2)	Stomata index (%)
N0	25.31 ± 4.99c	28.94 ± 4.15c	53.49 ± 3.73c	30.44 ± 4.15c	52.36 ± 4.19c	539.59 ± 55.04c	138.44 ± 11.50c	20.78 ± 4.41c
N1	27.90 ± 5.50b	32.89 ± 3.02b	57.40 ± 3.14b	34.39 ± 3.02b	55.90 ± 3.14b	584.11 ± 48.10b	149.22 ± 12.35b	26.11 ± 6.41b
N2	30.75 ± 7.02a	36.79 ± 1.89a	60.09 ± 2.87a	38.29 ± 1.89a	58.59 ± 2.87a	604.41 ± 47.01a	158.15 ± 11.47a	32.11 ± 6.82a
Mean	27.98 ± 6.23	32.87 ± 4.48	56.99 ± 4.22	34.37 ± 4.48	55.61 ± 4.26	576.03 ± 56.53	148.60 ± 14.17	26.33 ± 7.51
V1	21.74 ± 1.15d	32.28 ± 3.43abc	59.72 ± 1.80ab	33.78 ± 3.43abc	58.22 ± 1.80ab	622.00 ± 14.71b	154.78 ± 7.90bc	31.89 ± 7.83b
V2	23.30 ± 1.50d	33.63 ± 3.43abc	57.87 ± 3.41abc	35.13 ± 3.43abc	56.37 ± 3.41abc	591.78 ± 53.11c	151.00 ± 12.85d	30.56 ± 9.48b
V3	23.30 ± 1.21d	34.94 ± 2.44ab	54.56 ± 3.53c	36.44 ± 2.44ab	53.06 ± 3.53c	574.56 ± 40.98d	142.67 ± 9.96e	24.89 ± 6.97d
V4	36.22 ± 4.85a	30.06 ± 6.07c	56.49 ± 4.15bc	31.56 ± 6.07c	54.99 ± 4.15bb	513.67 ± 15.58f	133.00 ± 9.45g	19.22 ± 3.31f
V5	32.08 ± 1.54b	31.73 ± 5.40bc	58.14 ± 4.34abc	33.23 ± 5.40bc	57.76 ± 4.23ab	611.56 ± 15.81b	155.33 ± 5.20b	27.89 ± 2.98c
V6	26.17 ± 1.03c	30.13 ± 7.12c	54.02 ± 3.68c	31.63 ± 7.12c	52.52 ± 3.68c	500.33 ± 48.96f	137.44 ± 6.69f	21.11 ± 3.82e
V7	33.83 ± 1.71ab	35.95 ± 3.02a	55.37 ± 5.13c	37.45 ± 3.02a	53.87 ± 5.13bc	583.78 ± 30.12cd	152.56 ± 10.01cd	27.33 ± 4.80c
V8	32.76 ± 6.84b	32.96 ± 1.73abc	61.45 ± 2.35a	34.46 ± 1.73abc	59.95 ± 2.35a	647.00 ± 30.76a	171.56 ± 9.53a	34.78 ± 3.87a
V9	22.50 ± 4.16d	34.19 ± 2.40ab	55.33 ± 3.71c	35.69 ± 2.40ab	53.83 ± 3.71bc	539.67 ± 19.69e	139.11 ± 8.15f	19.33 ± 2.96f
Mean	27.98 ± 6.23	32.87 ± 4.48	56.99 ± 4.22	34.37 ± 4.48	55.61 ± 4.26	576.03 ± 56.53	148.60 ± 14.17	26.33 ± 7.51
N doses (n = 27)	**	**	**	**	**	**	**	**
Variety (n = 9)	**	**	**	**	**	**	**	**
N doses x Variety (n = 3)	**	**	ns	**	ns	**	**	**

DOI: 10.7717/peerj.18792/table-5

Note:

a, b; different letters in the same column indicate the difference between averages. V1, Alpu-2001; V2, Soyer-02; V3, Kate-A1; V4, Bezostaja-1; V5, Altay-2000; V6, Müfitbey; V7, Nacibey; V8, Harmankaya-99; V9, Sönmez-2001; Nitrogen doses, N0, Control; N1, 50 kg h⁻¹; N2, 100 kg ha⁻¹; **P ≤ 0.01; ns, non significant; sd, standard deviation.

Table 6:

Effects of treatment on flag leaf area and stomatal characteristics of wheat in 2021–2022 (2^nd year) growing season (N_n mean ± sd).

Nitrogen dose	Flag leaf area (cm2)	Abaxial stomata width (µm)	Abaxial stomata length (µm)	Adaxial stomata width (µm)	Adaxial stomata length (µm)	Epidermal cell (no./mm2)	Stomata density (no./mm2)	Stomata index (%)
N0	25.26 ± 5.04c	29.94 ± 4.15c	52.86 ± 4.19c	31.44 ± 4.15c	51.36 ± 4.19c	546.59 ± 55.04c	140.44 ± 11.50c	22.78 ± 4.41c
N1	28.05 ± 5.09b	33.89 ± 3.02b	56.40 ± 3.14b	35.39 ± 3.02b	54.90 ± 3.14b	591.11 ± 48.10b	151.22 ± 12.35b	28.11 ± 6.41b
N2	32.87 ± 8.07a	37.79 ± 1.89a	59.09 ± 2.87a	39.29 ± 1.89a	57.59 ± 2.87a	611.41 ± 47.01a	160.15 ± 11.47a	34.11 ± 6.82a
Mean	28.72 ± 6.91	33.87 ± 4.48	56.11 ± 4.26	35.37 ± 4.48	54.61 ± 4.26	583.03 ± 56.53	150.60 ± 14.17	28.33 ± 7.51
V1	21.54 ± 1.33e	33.28 ± 3.43abc	58.72 ± 1.80ab	34.78 ± 3.43abc	57.22 ± 1.80ab	629.00 ± 14.71b	156.78 ± 7.90bc	33.89 ± 7.83b
V2	22.62 ± 1.19e	34.63 ± 3.43abc	56.87 ± 3.41abc	36.13 ± 3.43abc	55.37 ± 3.41abc	598.78 ± 53.11c	153.00 ± 12.85d	32.56 ± 9.48b
V3	22.94 ± 1.02de	35.94 ± 2.44ab	53.56 ± 3.53c	37.44 ± 2.44ab	52.06 ± 3.53c	581.56 ± 40.98d	144.67 ± 9.96e	26.89 ± 6.97d
V4	35.39 ± 5.24a	31.06 ± 6.07c	55.49 ± 4.15bc	32.56 ± 6.07c	53.99 ± 4.15bc	520.67 ± 15.58f	135.00 ± 9.45g	21.22 ± 3.31f
V5	35.54 ± 2.53a	32.73 ± 5.40bc	58.26 ± 4.23ab	34.23 ± 5.40bc	56.76 ± 4.23ab	618.56 ± 15.81b	157.33 ± 5.20b	29.89 ± 2.98c
V6	25.81 ± 1.28d	31.13 ± 7.12c	53.02 ± 3.68c	32.63 ± 7.12c	51.52 ± 3.68c	507.33 ± 48.96f	139.44 ± 6.69f	23.11 ± 3.82e
V7	34.38 ± 5.82ab	36.95 ± 3.02a	54.37 ± 5.13bc	38.45 ± 3.02a	52.87 ± 5.13bc	590.78 ± 30.12cd	154.56 ± 10.01cd	29.33 ± 4.80c
V8	31.30 ± 7.48bc	33.96 ± 1.73abc	60.45 ± 2.35a	35.46 ± 1.73abc	58.95 ± 2.35a	654.00 ± 30.76a	173.56 ± 9.53a	36.78 ± 3.87a
V9	29.00 ± 7.48c	35.19 ± 2.40ab	54.33 ± 3.71bc	36.69 ± 2.40ab	52.83 ± 3.71bc	546.67 ± 19.69e	141.11 ± 8.15f	21.33 ± 2.96f
Mean	28.72 ± 6.91	33.87 ± 4.48	56.11 ± 4.26	35.37 ± 4.48	54.61 ± 4.26	583.03 ± 56.53	150.60 ± 14.17	28.33 ± 7.51
N doses (n = 27)	**	**	**	**	**	**	**	**
Variety (n = 9)	**	**	**	**	**	**	**	**
N doses x Variety (n = 3)	**	**	ns	**	ns	**	**	**

DOI: 10.7717/peerj.18792/table-6

Note:

a, b; different letters in the same column indicate the difference between averages. V1, Alpu-2001; V2, Soyer-02; V3, Kate-A1; V4, Bezostaja-1; V5, Altay-2000; V6, Müfitbey; V7, Nacibey; V8, Harmankaya-99; V9, Sönmez-2001; Nitrogen doses, N0, Control; N1, 50 kg ha⁻¹; N2, 100 kg ha⁻¹; **P ≤ 0.01; ns, non significant; sd, standard deviation.

As the N dose rate increased from 0 to 50 kg ha⁻¹, the flag leaf area increased from 25.31 to 27.90 cm² (10.23%), and similarly as the N dose rate increased from 50 to 100 kg ha⁻¹, the flag leaf area increased 10.21% at first year. In the second year, this ratio was determined between 11.04% and 17.18%, in relation to N doses respectively. The variation of flag leaf area index of the varieties was found between 21.74–36.22 cm² (first year) and between 21.54–35.54 cm² (second year) (Tables 5 and 6).

The highest flag leaf area (V4), N2 treatment (100 kg ha⁻¹) was obtained from variety x nitrogen interaction at both years (Figs. 5A, 6A).

Principal component analysis (pca)

A PCA was performed to explore the relationships among measured variables and assess how nitrogen doses (N0, N1, N2) influence the dataset of nine varieties of wheat. The PCA biplot revealed a clear separation among the nitrogen dose groups, reflecting differences in their multidimensional profiles.

The first and two dimensions (Dim1 and Dim2) accounted for a significant proportion of the total variance in the dataset, with Dim1 explaining 50.5% and Dim2 explaining 13.3%. Together, they captured the major trends within the data. According to the principal component analysis, the traits examined in the study were grouped into two main groups in terms of colour and length. The first group (Dim 1) consisted of seed yield (SY), plant height (PH), thousand grain weight (TGW), number of spikelets (NSS), number of spike (NS), number of grains (NGS), chlorophyll content (CLO), abaxial stomata width (ABSW), adaxial stomata width (ADSW) and flag leaf area (FLA) components that demonstrated strong positive intercorrelations. These features are associated with productivity and photosynthetic capacity, both of which are directly influenced by nitrogen availability. The second group (Dim 2) consisted of abaxial stomatal height (ABSH), adaxial stomatal height (ADSH), stoma density (SD), stoma index (SI) and epidermal cell number (ECN) that features represents stomatal and cellular characteristics. These components primarily aligned along Dim2 and has shown strong positive intercorrelations (Fig. 7). All the analyzed features showed nearly positive correlations while epidermal cell number (ECN) and flag leaf area (FLA) exhibited non correlation.

The nitrogen dose groups were distinctly separated along Dim1 and Dim2. The control group (N0) clustered toward the negative end of Dim1, suggesting lower values of variables strongly associated with this dimension. This indicates a limited growth response in the absence of nitrogen. In contrast, the 100 kg ha⁻¹ N dose group (N2) positioned toward the positive end of Dim2, indicating higher growth and yield parameters. The 50 kg ha⁻¹ N dose group (N1) occupied an intermediate position, bridging the control and 100 kg ha⁻¹ N groups, showed moderate increases in features (Fig. 8). Interestingly, stomatal traits (Dim2) show less dramatic shifts across N levels, suggesting that these features are less directly affected by N dosage. The results highlight the dose-dependent effects of N on plant development and productivity parameters.

The color gradient in Fig. 9 represents the contribution of each variety to the principal components. Varieties with higher contributions, such as Bezostaja-1 (V4) and Sönmez- 2001 (V9), are more influential in defining the axes of variation. This highlights the importance of varietal differences in the effectiveness of nitrogen application.

Discussion

There is a relationship between the N dose and the yield (Paul et al., 2017; Luo et al., 2019). As we found in the present study, seed yield and agronomic traits had positive relations with N dose treatments. Many studies have reported that increase of N also increase yield and agronomic traits (Fang et al., 2018; Lollato et al., 2019; Liu, Liao & Liu, 2021). In our study, as N doses increased, all yield and morphological parameters increased as well. High N dose stimulates the development of vegetative organs and reduces the amount of assimilation carried to the spikes (Sun et al., 2019). While the high values obtained in the examined properties were obtained from the highest N dose, the percentage of increase occurring in the increase from control (0 kg ha⁻¹) to 50 kg ha⁻¹ was greater than the percentage of increase occurring in the increase from 50 to 100 kg ha⁻¹. The highest seed yield was obtained from 100 kg ha⁻¹ nitrogen application in our study. Kayan et al. (2020) determined 100 kg ha⁻¹ as the most suitable N dose in terms of seed yield and N efficiency among N doses. The rapid response of seed yield to N increases at low N levels is due to the increase in tiller formation and the resulting increase in seed number when the soil N amount is insufficient for maximum yield (Hoogmoed et al., 2018). Despite the potential increase in plant N concentration, subsequent slow growth phases indicate that increases in N uptake per plant beyond the maximum yield response point result in low improvement in yield per plant (Xu et al., 2018). Different N dose could improve the photosynthetic capacity by regulating the plant height, thereby increasing production (Lollato et al., 2019). As a result of the correlation, it was determined that seed yield was highly positively correlated with plant height, number of spikelets per spikes, number of seeds per spike and 1,000 seeds weight with increase N treatment in both years in our study (Table 2). Xue et al. (2006) reported that N dose improved yield by increasing seed number. That the improving number of spikes and number of seeds per spike may be the primary factors for increasing yield (Si et al., 2020). Saleem et al. (2010) stated that more studies are required to identify wheat cultivars that maintain high seed yield with lower N dose requirements. That researchers found seed yield increased significantly and progressively with each increment of N from 0 to 150 kg ha⁻¹, further addition of N above 150 kg ha⁻¹ did not lead to additional increase, but rather decrease in seed yield ha⁻¹.

According to our results, chlorophyll content in the flag leaf varied among N doses and growth times. While chlorophyll content increased as N doses increased, chlorophyll values were found higher in early growth period than from late growth periods. N, which is found in the structure of chlorophylls that play a role in photosynthesis, is very important for chlorophylls (Zangani et al., 2021). Most of the N is found in the leaf in chloroplasts and mostly in ribulose bisphosphate carboxylase (Makino et al., 1997). Therefore, in case of N deficiency, there will be a decrease in rubisco activity and a decrease in photosynthesis. Photosynthesis plays a direct role in the growth, development and yield of plants. Therefore, changes in pigments that play a role in photosynthesis mean changes in the rate of photosynthesis in plants (Sallam et al., 2019). As a result, seed yield decreases (Tóth et al., 2002). The amount of chlorophyll varies between species and may also vary within a species. In addition, leaf structure is one of the important factors that determine the amount of chlorophyll (Criado et al., 2007). In our study the highest chlorophyll content was found of Bezostaja-1 and Nacibey wheat varieties (V4 and V7, respectively) both of years due to the highest flag leaf area. The excess chlorophyll content in plants stimulates growth and development while increasing yield. The study results confirm the data and draw attention to the role of N in yield increase (Sharifi & Mohammadkhani, 2016). In addition, the chlorophyll content of flag leaves in grasses can provide important clues about the growth and development status of plants (Qian et al., 2021; Pandey et al., 2023). Also, the amount of chlorophyll in plants varies depending on time during the vegetation period (Čaňová, Ďurkovič & Hladká, 2008) and it is closely related to plant photosynthetic capacity (Zhang et al., 2022), N is transferred from leaves to spikes, resulting in a decrease in spectral reflectance and chlorophyll content (Zavoruev & Zavorueva, 2003). Shirazi et al. (2014) reported that leaf area is an important indicator of crop growth. Biomass production in plants is closely related to leaf area (Man, Yu & Shi, 2017). Li et al. (2023) stated that the leaf area increases, the amount of biomass and seed yield also increases. While the leaf area was 25.28 cm² at the control dose, it increased to 31.80 cm² at the 100 kg ha⁻¹ dose. This showed an increase of 25.79% in the leaf area. The increase in biomass production will play an important role in improving the yield of wheat, and increased biomass production should be given priority to increase seed production of wheat (Li et al., 2023). In our study, an increase in leaf area was observed with increasing N doses, thus an increase in yield was observed.

Chlorophyll content in flag leaf starts to decrease with time after antesis, regardless of treatments and varieties, as plants undergo natural senescence during seed filling (Kumar et al., 2022). Steer & Harrigan (1986) reported an average decrease of 23% in chlorophyll content from antesis to physiological maturity (Zhao et al., 2005). In line with previous studies, our research findings indicate a decrease in chlorophyll content as the growth stages is delayed.

Decreases in chlorophyll content may mark the start of senescence (Bassi, Menossi & Mattiello, 2018). Indeed, in studies conducted in sugar cane, total chlorophyll content was found to be higher in young leaves and with increased N treatments (Bassi, Menossi & Mattiello, 2018). In our study, 1st growth stages (GS1) of wheat was found the highest chlorophyll content.

In addition, parameters such as the position of stomata on the leaf (being lower or upper) and stomata density, which play an active role in gas exchange parameters, are parameters that increase the functionality of the chlorophyll in photosynthesis (Wasaya et al., 2021). Therefore, the reason for the yield differences between varieties can be associated with changes in these parameters.

Stomata of flag leaf are an effective property in the compatibility of varieties to different environmental conditions, which can have an important role in gaining high performance. However, this effectiveness can be affected by environmental conditions and different growing seasons (Anjum et al., 2011; Limochi & Eskandari, 2013; Caine et al., 2019). The stomatal traits help plants maintain photosynthetic rates under water deficit (Ding et al., 2018) and high temperature (Reynolds-Henne et al., 2010; Farooq et al., 2011; Sharma et al., 2015; Urban et al., 2017; Caine et al., 2023). The open stomata maintain adequate gas exchange for photosynthesis, which is vital for plant metabolism (Sayed, 2003; Reynolds-Henne et al., 2010; Sharma et al., 2015; Tricker et al., 2018; Zhao et al., 2020). With increased photosynthesis, plant productivity increases. However, depending on the plant species, one or more of the same stress factors may have different effects on stomata (Merilo et al., 2014; Sharma et al., 2015; Sommer et al., 2023) and, stomata density and size in cultivated wheat tend to be larger and sparser than in wild species (Wang et al., 2022).

The plants increase the number of stomata to minimize transpiration and water loss and closing stomata (Yavas et al., 2024). The researcher stated that planting date and varieties were found significant effects on all anatomical properties including stomata area, stomata diameter and stomata number of rice. Early planting (22 May) reduced the area and diameter of stomata, also grain yield. Grain yield showed maximum correlation with stomata area (0.405). Researchers suggested that this property can be used as an important factor in breeding programs for improving rice grain yield (Limochi & Eskandari, 2013).

In our study, all stomatal parameters increased with increasing N doses (Tables 5 and 6). and among the wheat varieties, the highest stomatal index and density were obtained from Harmankaya-99 wheat variety (V8) (Figs. 5E and 6E) in both years. It has been determined that the criteria considered in this study, together with N doses, show significant effects among varieties, and therefore yield increases are achieved at significant levels.

The effect of N fertilizer applications on stomatal conductance, flag leaf area and other physiological parameters of sorghum was found to be insignificant (Bruns, 2016). In another study, the N fertilizer increased, stomatal conductance of rapeseed increased as much as 21%, and it was not statistically significant for rapeseed (Nasab et al., 2014).

The flag leaf is the main component of the canopy in growth stages of wheat, and it determines the grain-filling rate and the yield (Vicente et al., 2018). Some studies have shown that the number and density of stomata significantly affect their ability to regulate photosynthesis and transpiration. The stomatal density of wheat flag leaves increased under limited water irrigation, and later irrigation resulted in greater stomatal density. Also, the stomatal length and width decreased, stomatal density of plant leaves increased and the stomata got smaller under stress conditions, but changes in the stomatal shape varied greatly (Bertolino, Caine & Gray, 2019; Liu, Liao & Liu, 2021).

In the present study, according to the principal component analysis, PCA biplot that visualizes the relationships between different varieties of a particular dataset. The x-axis represents the first principal component (Dim1), which accounts for 50.5% of the variance in the data, while the y-axis represents the second principal component (Dim2), which accounts for 13.3% of the variance. The data points, representing the different varieties, are colored and sized according to their contribution (Contrib) and variety (Variety), respectively. Varieties that are closer together on the plot are more similar to each other, while those that are farther apart are more distinct. The arrows indicate the direction and magnitude of the correlation between the principal components and the original variables. Varieties with longer arrows have a stronger influence on the principal components. This PCA biplot provides a useful overview of the underlying structure of the dataset, allowing the researcher to identify patterns, clusters, and relationships between the different varieties. The information gleaned from this analysis can inform further research, product development, or other desicion-making processes. Koppensteiner et al. (2022) stated that for experimental data at 0 g N m-2, PC1 explained 55.3% experimental data of all N fertilization rates, PC1 carried 51.5% and PC2 28.9% of variation summing up to 80.4%. Similarly, Zewdu, Mekonnen & Geleta (2024) were the first four principal components accounted for 76.76% of the total observed phenotypic variation at the results of principal component analysis. Tunç, Yılmaz & Yaman (2023) stated that in their research on the olive varieties that the first five components consisted of 79.08% of the total variation in terms of stomatal morphology and chlorophyll content.

As researchers have stated that principal component analysis of yield and yield components in wheat generally show highly varying results which can be attributed to varying experimental and environmental conditions (Ozukum et al., 2019; Nayana, Kumar & Chesneau, 2022; Bonfil et al., 2023).

Conclusions

This research has confirmed the hypothesis that nitrogen is an effective element on growth, development, productivity, chlorophyll content, abaxial stomata width, abaxial stomata length, adaxial stomata width, adaxial stomata length, epidermal cells, stomata cells, stomata density and stomata index in plants. In this 2-year study, the increase in N dose boosted the number of spikelets per plant, the number of seeds per spike and 1,000 seed weight, and accordingly, seed yield per hectare increased. In addition, as the leaf area index increased, so did the chlorophyll amount and stomata size and thus photosynthetic efficiency increased and accordingly, the amount of matter accumulated in the seed increased. It was determined that the best results in yield and other parameters examined were obtained from the 100 kg ha⁻¹ N dose. The hypothesis that nitrogen doses significantly boosted abaxial stomata width, abaxial stomata length, adaxial stomata width, adaxial stomata length, epidermal cell, stomata density, stomata index and stomata density was confirmed in this study. Significant increases were obtained with nitrogen doses in flag leaf area, which plays a role in photosynthetic efficiency. Yields were higher in varieties with denser stomata, while yields were lower in varieties with less dense stomata. More detailed studies should be conducted based on the results of this study to gain a deeper understanding of the relationships between stomatal densities and N fertilization, and thus yield increase.

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