Influence of phosphorous fertilizer on mineral nutrition and yield attributes of wheat: Acquisition with PSTOL1 gene for arid environment

2.1 Growth and development characteristics

The different growth and development parameters including plant height, leaf area index, heat unit efficiency and SPAD were recorded at vegetative stage.

1. Plant height was measured as a distance in centimeter from the base to the tip of spike excluding the awns at the physiological maturity stage.

2. Leaf area index was measured from randomly selected five flag leaves according to techniques of (Ahmad et al., 2015)

1. Heat-use efficiency (HUE) [kg ha⁻¹ °C/day] was recorded according (Nandini & Sridhara, 2019) technique

1. SPAD value/ total chlorophyll contents were measured using portable chlorophyll meter “SPAD-502 DL Meter” (Minolta Camera co. Ltd. Osaka, Japan)

2.2 Yield

The wheat crop was harvested at its physiological maturity stage (when the green colour from the glumes and kernels had disappeared completely) during 2nd week of April. The grain yield and its determinants; number of productive tillers; spike length; number of spikelets spike^-1; 1000 grain weight; number of grains spike^-1; straw yield, grain yield and harvest index were quantified by employing standard protocols.

2.3 Protein, fat and ash content

The protein, fat and ash contents in wheat grains were calculated by (Rehman et al., 2020) methodology using following equations. $$Protein\left(\%\right)=N_2\left(\%\right)\times 6.25$$

$\mathit{Fatcontents}(\%)=\frac{\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{b}\mathrm{e}\mathrm{a}\mathrm{k}\mathrm{e}\mathrm{r}\mathrm{w}\mathrm{i}\mathrm{t}\mathrm{h}\mathrm{f}\mathrm{a}\mathrm{t}\mathrm{t}\mathrm{y}\mathrm{m}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{a}\mathrm{l}(\mathrm{g})-\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{e}\mathrm{m}\mathrm{p}\mathrm{t}\mathrm{y}\mathrm{b}\mathrm{e}\mathrm{a}\mathrm{k}\mathrm{e}\mathrm{r}(\mathrm{g})}{\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{o}\mathrm{r}\mathrm{i}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{l}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}(\mathrm{g})}\times 100$ $$\mathit{Ashcontents}(\%)=\frac{\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{a}\mathrm{f}\mathrm{t}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{s}\mathrm{h}\mathrm{i}\mathrm{n}\mathrm{g}(\mathrm{g})}{\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{o}\mathrm{r}\mathrm{i}\mathrm{g}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{l}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}(\mathrm{g})}\times 100$$

2.4 Ionic Constituents

The N, P, K, Zn and Fe contents in wheat grains and straw was analyzed after digestion of said plant material in con. H₂SO₄. The N concentration was determined by Kjeldahl method (Method 7.1.3) and P by colorimetry (ammonium vanadate-ammonium molybdate [Method 7.2]. The contents of K, Zn, and Fe were analyzed by wet digestion method (Ryan et al., 2001; Ryan et al., 2009).

2.5 Nutrients-use-efficiency

The different components of nutrient-use-efficiency; partial factor productivity, internal use efficiency, partial nutrient budget and agronomic efficiency (Sarkar et al., 2021; Rawal et al., 2022) for applied P-fertilizers in wheat crop was calculated at after harvest.

2.6 Economic analysis

The partial budget analysis was carried out, according to open marketing of fertilizers and grain produces at the time of planting and harvesting. The marginal rate of return (MRR) was measured according to protocol proposed by (Byerlee & Siddiq, 1994). $$\mathit{Ashcontents}(\%)=\frac{\mathrm{C}\mathrm{h}\mathrm{a}\mathrm{n}\mathrm{g}\mathrm{e}\mathrm{i}\mathrm{n}\mathrm{N}\mathrm{B}(\mathrm{N}\mathrm{B}\mathrm{b}-\mathrm{N}\mathrm{B}\mathrm{a})}{\mathrm{C}\mathrm{h}\mathrm{a}\mathrm{n}\mathrm{g}\mathrm{e}\mathrm{i}\mathrm{n}\mathrm{T}\mathrm{V}\mathrm{C}(\mathrm{T}\mathrm{V}\mathrm{C}\mathrm{b}-\mathrm{T}\mathrm{V}\mathrm{C}\mathrm{a})}\times 100$$ Where; NB = net profit (ha⁻¹) for each treatment [difference between the gross field benefit (GFB) and total variable cost (TVC); TVC = total variable cost is the sum of expenses incurred on costs and application of fertilizer in the field.

2.7 RNA synthesis, DNA extraction and qPCR analysis

To investigate the expression of PSTOL1 at 80 Kg P₂O₅ ha^-1P-fertilizer level in wheat varieties, the mature leaves were harvested and RNA extracted for advanced analysis. PSTOL1 sequences, originally isolated from Oryza sativa, were acquired from the NCBI database for BLAST analysis, excluding duplicate entries and retaining sequences that exhibited over 50 % identity. Complementary DNA was synthesized employing a Thermos Scientific Revert Aid Reverse Transcriptase kit, and primers were crafted using Beacon designer software. The expression levels after P-fertilization was quantified via real-time quantitative PCR (Applied Biosystems® 7900 HT Fast RT-PCR), with Actin serving as the reference gene. Expression quantification was conducted employing the 2^-ΔΔCT method (Ma et al., 2006).

2.8 Statistical analysis

The data was analyzed statically using computer-based software COSTAT (6.311, USA). To evaluate the relationships among the studied traits, we employed multivariate analysis (PCA via biplot), correlation matrix (with ggplot2), and constructed a heatmap using customized code (heatmap) within the R statistical software.

3.1 Physiological and morphological parameters

An increase in plant height of wheat varieties was observed after application of different doses of Phosphorous (P) fertilizer. The increase in plant height at 40 Kg P₂O₅ ha⁻¹ was 5 % in Anaj-2017; at 80 Kg P₂O₅ ha⁻¹ this enhancement was maximum of all doses; 11 % in Zincol-2016, Akbar-2019 and FSD-2008, while at 120 Kg P₂O₅ ha⁻¹ highest increase was 9 % in Anaj-2017 (Fig. 1).

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Fig. 1

The Impact of Different Phosphorus Fertilizer Doses (40, 80, and 120 kg P2O5 per hectare) on Four Wheat Varieties (Zincol-2016, Akbar-2019, FSD-2008, and Anaj-2017) in Terms of (A) Plant Height (cm), (B) SPAD (Soil Plant Analysis Development) Values, (C) Leaf Area Index, and (D) Heat Unit Efficiency (HUE) (kg per hectare per °C Day). R = Replication; V = Varieties; T = Phosphorus Fertilizer Doses; *** = Highly Significant at P ≤ 0.001; ** = Significant at P ≤ 0.01; ns = Not Significant.

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Total leaf chlorophyll contents (SPAD) were remarkedly enhanced in all wheat varieties after application of different doses of P-fertilizer. The increase in SPAD at 40,80, and 120 Kg P₂O₅ ha⁻¹ was observed to 8 %, 21 % and 15 % respectively in wheat variety Akbar-2019 (Fig. 1).

The application of different doses of P-fertilizer affected LAI in all wheat varieties. The maximum increase in LAI over control at 40, 80, and 120 Kg P₂O₅ ha⁻¹ was observed to 13 %, 30 % and 34 % respectively in wheat variety FSD-2008.

Application of Phosphorous fertilizer affected heat unit efficiency (HUE) in almost all wheat varieties. As compared to control maximum HUE at 40,80, and120 Kg P₂O₅ ha⁻¹ was recorded to 8 %, 21 % and 15 % respectively in wheat variety Akbar-2019 (Fig. 1).

3.2 Yield

The number of productive tillers of wheat varieties were remarked enhanced after application of P₂O₅ fertilizer. As compared to control plants maximum increase (11 %) in productive tillers was observed in Zincol-2016 at 80 Kg P₂O₅ ha⁻¹ fertilizer application level. However, at the same fertilizer level Anaj-2017 showed 10 % increase in productive tillers as compared to control (Table 1). As compared to control plants maximum increase (12 %) in spike length was observed in Zincol-2016 at 80 Kg P₂O₅ ha⁻¹ fertilizer level. However, at the same fertilizer level FSD-2008 showed 10 % increase in spike length as compared to control (Table 1).

A maximum enhancement in the number of spikelet’s spike^-1 was observed at 80 Kg P₂O₅ ha⁻¹ application. As compared to un-fertilized wheat plants, an increase of 9 %, 13 %, 10 % and 8 % was recorded in Zincol-2016, Akbar-2019, FSD-2008 and Anaj-2017 respectively. However, at 120 Kg P₂O₅ ha⁻¹ fertilizer level, Akbar-2019 showed 9 % increase in number of spikelet’s spike^-1 (Table 1). An increase in 1000 grain weight was observed after application of P-fertilizer in all wheat varieties. As compared to control maximum increase of 16 % and 12 % was recorded in Akbar-2019 and Zincol-2016 respectively at 80 Kg P₂O₅ ha⁻¹ (Table 1).

Higher enhancement in number of grains spike^-1 was observed after application of P-fertilizer. At 80 Kg P₂O₅ ha⁻¹, maximum enhancement in number of grains spike^-1 was observed in Akbar-2019 (14 %) and Zincol-2016 (11 %) (Table 1). A slight change in straw yield was observed in all wheat varieties after application of P₂O₅ fertilizer. As compared to control 4 % increase in straw yield was observed in Zincol-2016, Akbar-2019 and FSD-2008 at 80 Kg P₂O₅ ha⁻¹ (Table 1). The application of P-fertilizer enhanced grain yield of wheat varieties. As compared to control plants highest grain yield (21 %) was observed in Akbar-2019 at 80 Kg P₂O₅ ha⁻¹. However, at the same fertilizer level Zincol-2016 showed 13 % increase in grain yield as compared to control. The data showed that as compared to unfertilized wheat plants, highest harvest index (HI) at 40 Kg P₂O₅ ha⁻¹ was observed in Zincol-2016 (5 %), at 80 Kg P₂O₅ ha⁻¹ Akbar-2019 (11 %) and at 120 Kg P₂O₅ ha⁻¹ Zincol-2016 (3 %) (Table 1).

3.3 Ionic constituents

The application of P-fertilizer enhanced the uptake of N-contents in straw and grains of all wheat varieties. In Zincol-2016 as compared to control plants, 8 %, 11 % and 13 % straw N-contents were observed at 40, 80 and 120 Kg P₂O₅ ha⁻¹ respectively (Table 2). Similarly at 40 Kg P₂O₅ ha⁻¹ maximum N-contents (22 %) in grains were observed in Anaj-2017 while at 80, and 120 kg P₂O₅ ha⁻¹ this was 42 % and 63 % respectively in Zincol-2016. As compared to control the maximum increase in straw P-contents at 40, 80 and 120 Kg P₂O₅ ha⁻¹ was observed in Anaj-2017 as 36 %, 35 % and 95 % respectively. However, grain P-contents at 40 Kg P₂O₅ ha⁻¹ were higher in FSD-2008, while at 80 and 120 kg P₂O₅ ha⁻¹ this increase was highest 64 % and 92 % respectively in Zincol-2016 compared to control (Table 2). The uptake of straw and grains of K-contents was remarkably increased after application of P-fertilizer in all wheat varieties. As compared to control plants maximum straw K-contents were recorded in FSD-2008, 33 %, 69 % and 79 % at 40, 80 and 120 Kg P₂O₅ ha⁻¹ respectively. On the other hand, as compared to non-fertilized wheat plants, maximum increase in grain K-contents at 40, 80 and 120 Kg P₂O₅ ha⁻¹ was recorded in Zincol-2016, 11 %, 17 % and 28 % respectively (Table 2).

Zn-contents in straw and grains of all wheat varieties were remarked reduced after application of P-fertilizer (80 and 120 Kg P₂O₅ ha⁻¹). At 40 Kg P₂O₅ ha⁻¹ the Zn straw and grain contents were enhanced to 22 % in Anaj-2017 and Zincol-2016 respectively. While as compared to control plants maximum decrease (23 %) in Zn straw contents was observed in Akbar-2019 at 120 P₂O₅ ha⁻¹. Similarly, Zn-grain contents were reduced to 19 % at 120 Kg P₂O₅ ha⁻¹ in FSD-2008 and Anaj-2017 (Table 2). Data showed that Fe-contents in straw and grains were also reduced after application of P-fertilizer (80 and 120 kg P₂O₅ ha⁻¹), while enhanced at 40 kg P₂O₅ ha⁻¹. As compared to control the maximum increase in P-contents was observed in Akbar-2019 (9 % and 11 %) in straw and grain respectively at 40 kg P₂O₅ ha⁻¹. However, at 80 and 120 kg P₂O₅ ha⁻¹, Fe-contents in straw and grain were reduced to 11 % and 19 % respectively in Akbar-2019. Similarly, grain Fe-contents were lowered to 9 % and 18 % in FSD-2008 at 80 and 120 Kg P₂O₅ ha⁻¹ respectively (Table 2).

3.4 Protein, fat and ash content

Data for wheat grain protein, fat and ash contents differed statistically in response to application of P_-fertilizer. As compared to non-fertilized plants an increase in grain protein contents to 16 % and 13 % at 40 and 120 Kg P₂O₅ ha⁻¹ respectively was recorded in Akbar-2019, however at 80 Kg P₂O₅ ha⁻¹ maximum increase (11 %) was observed in FSD-2008 (Table 3). At 40 Kg P₂O₅ ha⁻¹ maximum increase (10 %) in fat contents was observed in Zincol-2016, however at 80 and 120 Kg P₂O₅ ha⁻¹, FSD-2008 achieved maximum 12 % and 23 % grain fat contents respectively as compared to un-treated plants. It was observed that as compared to other wheat varieties, the Anaj-2017 produced higher (5.9 %) grain ash contents at 40, 80, 120 Kg P₂O₅ ha⁻¹ respectively (Table 3).

3.5 Phosphorus nutrient use efficiency

The application of P-fertilizer significantly enhanced nutrient use efficiency of wheat varieties. As compared to control, Anaj-2017 exhibited maximum 301 % followed by FSD-2008 (280 %), Zincol-2016 (264 %) and Akbar-2019 (258 %) partial factor productivity (PFP) at 80 Kg P₂O₅ ha⁻¹ (Fig. 2). The maximum internal use efficiency (IUE) was recorded as 91 %, 99 % and 70 % at 40, 80 and 120 Kg P₂O₅ ha⁻¹ respectively in Anaj-2017 (Fig. 2). A remarked increase in agronomic efficiency (AE) was observed in in Anaj-2017 (245 %) at 80 Kg P₂O₅ ha⁻¹ fertilizer level as compared to control. Similarly, at the same fertilizer level Akbar-2019 (240 %), Zincol-2016 (237 %) and FSD-2008 (232 %) also showed better AE as compared to their respective controls and other two P-fertilizer levels (40 and 120 Kg P₂O₅ ha⁻¹) (Fig. 2). At 80 Kg P₂O₅ ha⁻¹ a maximum value for partial nutrient budget (PNB) was recorded in Anaj-2017 (379 %) followed by FSD-2008 (385 %), Zincol-2016 (342 %) and Akbar-2019 (335 %) (Table 4).

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Fig. 2

Influence of Various Phosphorus Fertilizer Doses (40, 80, and 120 kg P2O5 per hectare) on Four Wheat Varieties (Zincol-2016, Akbar-2019, FSD-2008, and Anaj-2017) Regarding (A) Agronomic Efficiency, (B) Internal Use Efficiency, (C) Partial Factor Productivity, and (D) Partial Nutrient Budget (kg per kg).

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3.6 Economic analysis

For economic analysis all the input costs excluding P-fertilizer were bought from market. Grain yield was settled to 12.50 % downward to reduce farm management losses during experimental yield and farmer’s yield. The results uncovered that marginal rate of return (MRR) by P-fertilizer application was minimum in FSD-2008 (125.3 %) at 120 kg P₂O₅ ha⁻¹ while maximum in Akbar-2019 (241.7 %) at 80 kg P₂O₅ ha⁻¹. Among all wheat varieties Akbar-2019 was more efficient in utilizing phosphorus nutrient and had better marginal benefit over cost (PKR 52,200 ha⁻¹) at 80 kg P₂O₅ ha⁻¹ (Table 4).

3.7 Multivariate analysis

The traits studied under P-fertilizer showed a significant correlation among different studied parameters. The pH was strongly correlated with GY, NPT, NGSA, HI, SPL, and SY, while the SPAD was correlated with HUE and PH. The HI was remarked and positively correlated with SPL, SY, and TGW. Ash, Pro, and Fat were positively correlated with SNC, GNC, SPC, GPC, SKC, and GKC, whereas PFP, AE, and PNB were correlated with IUE. A significant negative correlation was observed among GNC, SKC, GKC, SPC, SNC, PFP, PNB, and AE (Fig. 3).

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Fig. 3

Correlation among (A) growth and yield parameters, (B) ionic contents, nutritional components and phosphorus nutrient use efficiency of four wheat varieties (Zincol-2016, Akbar-2019, FSD-2008 and Anaj-2017) grown under influence of different doses of P-fertilizer (40, 80 and 120 Kg P₂O₅ ha⁻¹). Ash-ash, Pro-protein, Fat-fat, GNC-grain nitrogen content, SNC- straw nitrogen content, SKC-straw potassium content, GKC- grain potassium content, GPC-grain phosphorus content, SPC- straw phosphorus content, GFC-grain iron content, SFC-straw iron content, GZC-grain zinc content, SZC-straw zinc content, AE- agronomic efficiency, IUE- internal use efficiency, PFP- partial factor productivity, PNB- partial nutrient budget, GY-grain yield, SY-straw yield, PH-plant height, SPAD-soil plant analysis development, LAI-leaf area index, HUE-heat unit efficiency, NPT-number of productive tillers, SPL-spike length, NSP-number of spikelet’s per spike, TGW-1000 grain weight, NGS-number of grains per spike, HI-harvest index.

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3.8 Gene expression analysis

The study examined the expression profile of PSTOL1 of two wheat varieties at 80 Kg P₂O₅ ha⁻¹. The PSTOL1 exhibited a significant increase in expression compared to the control after application of P-fertilizer. As compared to control, 217 %, 129 %, 100 % and 93 % increase was observed in Zincol-2016, Akbar-2019, FSD-2008 and Anaj-2017 respectively (Fig. 4).

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Fig. 4

Relative expression levels of PSTOL1 in in leaves of wheat varieties after application of 80 Kg P₂O₅ ha⁻¹.

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