3.2.4. Effect of Combined Stress on Yield of the Wheat Plant

Table 1 shows the effect of combined stress on the yield of the wheat plant. The crop yield kg ha−<sup>1</sup> was reduced significantly (*<sup>p</sup>* ≤ 0.05) from 10,270 in the control group to 2309.10 in the stressed one. The 1000-grain weight was significantly (*p* ≤ 0.05) reduced from 56.75 g in control to 22.80 g in stress conditions.

The correlation matrix (Table 2) shows the negative correlation between spike number and grain weight. The leaf area, photosynthesis, SPAD, tiller, and spike weight were positively correlated. The PCA matrix and plot (Table 3 and Figure 8) show the spike length, spike weight, and leaf area as the most prominent parameters affecting plant yield once the leaf area was reduced, which might have been due to the waterlogging stress. As a result, photosynthesis was reduced, and ultimately, spike weight was compromised. On the other hand, once spike weight was compromised, ultimately, grain weight was reduced, and finally, the plant yield was reduced.


 plant height; T: tillers (FS); LA: leaf area (FS); SPAD: SPAD (15DAFS); S.P: spikes/plant; SL: spike length; SW: spike weight; S.S: spikelet/spike; GW.S: grain weight/spike; G.S: grains/spike; IGW: individual grain weight; TGW: thousand-grain weight; GY.P: grain yield/plant; B.P: biomass/plant; HI: harvest index; Ymann.ac: yield (Mann/acre); Ykg.ha: yield (kg ha−1); FS: flowering stage; DAFS: days after flowering stage.

PH:

**Table 2.**

Correlation

 between

morphological

 attributes of the wheat plants grown under abiotic stresses.


**Table 3.** Principal component analysis between morphology and abiotic stress responses.

Note: PH: plant height; T: tillers (FS); LA: leaf area (FS); SPAD: SPAD (15DAFS); S.P: spikes/plant; SL: spike length; SW: spike weight; S.S: spikelet/spike; GW.S: grain weight/spike; G.S: grains/spike; IGW: individual grain weight; TGW: thousand-grain weight; GY.P: grain yield/plant; B.P: biomass/plant; HI: harvest index; Ymann.ac: yield (Mann/acre); Ykg.ha: yield (kg ha−1); FS: flowering stage; DAFS: days after flowering stage.

**Figure 8.** PCA plot showing chief components of plant stress responses. Note: PH: plant height; T: tillers (FS); LA: leaf area (FS); SPAD: SPAD (15DAFS); S.P: spikes/plant; SL: spike length; SW: spike weight; S.S: spikelet/spike; GW.S: grain weight/spike; G.S: grains/spike; IGW: individual grain weight; TGW: thousand-grain weight; GY.P: grain yield/plant; B.P: biomass/plant; HI: harvest index; Ymann.ac: yield (Mann/acre); Ykg.ha: yield (kg ha<sup>−</sup>1); FS: flowering stage; DAFS: days after flowering stage.

#### 3.2.5. NPK in the Plant and Stress Response

Fertilizer is an essential resource for plant growth. The results of NPK in wheat plants grown under different abiotic stresses and with SCU application are shown in Table 4. It was revealed that NPK was highest in control and lowest in the case of heat and combined stresses. At the heat stress application stage, the plant was already at the flowering stage, and %N was significantly (*p* ≤ 0.05) reduced from 2.8 to 1.8%, P was reduced from 0.31 mg/kg to 0.24 mg/kg, and %K was reduced 1.2 to 0.98%. A 20% decrease in %K was observed in waterlogging stress, and it was the same in all treatments. Combined stressed accumulated the least nitrogen (1.33%) and P (0.98%) in plants among all treatments (Table 4).

**Table 4.** Phosphorous, potassium, and nitrogen accumulation in wheat plants grown under different abiotic stresses.


Note: lowercase letters show the significant differences among treatments according to one-way ANOVA and DMRT at a significance level of *p* ≤ 0.05.

#### **4. Discussion**

In the current experiment, the effect of different abiotic stresses was analyzed individually and in a combined form under the influence of SCU. It was found that the spike weight and root network were affected by all the stresses, but heat stress significantly (*p* ≤ 0.05) reduced crop yield (Table 1). Our results of crop yield reduction due to heat stress were following the previous studies conducted on wheat and soya bean [7–9]. It was also found that the nitrogen release period was key to the stress alleviation for the plant at a growth point of ~120 days (that is, nitrogen release period of the fertilizer), as the plant was able to grow in all stressed environments. However, after this phase (at maturity and late flowering), plant growth was significantly (*p* ≤ 0.05) reduced. Our studies observed correlations between growth parameters, such as spike number and grain weight leaf area, photosynthesis, SPAD, tiller, and spike weight (Table 2). Studies in the past also revealed that different growth parameters were related to each other, and changes in one growth parameter could cause alterations in the other parameters of the wheat plant [32]. The PCA matrix and plot (Table 3 and Figure 8) show the spike length, spike weight, and leaf area as the most prominent parameters affecting crop yield. Many studies have suggested the dry weight of wheat seedlings as the best principle for measuring the stress resistance attribute of the genotype [33,34]. A further detailed discussion of these results is given below.

The stress (waterlogging, salt stress, and heat) tolerance in the wheat plants is a matter of growth tolerance in plants, which initiate vigorous root systems and proliferate abundantly [1,2,33]. Our experiments also showed fewer tillers and reduced photosynthetic rate, fluorescence, dry biomass, leaf area, SPAD value, and crop yield, especially for the heat and salt stresses. However, these effects were moderate in waterlogging stress, especially for the first 120 days (Table 1, Figures 2–4 and 6). Salt stress was already reported to adversely affect plants' growth by Yu et al. [10]. In another study, Domico et al. [11] also reported reduced metabolic activity in the cardoon plant when subjected to long-term and short-term salt stress. The work of Pezo et al. further supported our results [12], reporting the reduced crop yield and pepper seed quality under salt stress.

Similarly, our results of reduced photosynthetic rate, leaf area, dry biomass, and yield were consistent with previous research conducted on wheat [7] and soya bean [8]. Waterlogging is undoubtedly reported to negatively impact a plant's physiological and biochemical response [16–18]. However, few studies also reported the innate adaptability of a plant against waterlogging stress [35]. It might be attributed to the capability of a plant to grow in hypoxic conditions that are mainly based on the root system tolerance of a plant [18,35]. It was hypothesized that the Yangmai 25 was able to build a good root network. That is why it was less affected by waterlogging stress than other stresses. In

the first phase of waterlogging stress, the root growth might have been rapid or close to that of the control plants in Yangmai 25, but after ~120 days, the growth of the roots might have been compromised, which resulted in the decreased crop yield (Table 1). Once the root growth is clasped, the shoot growth can never recover to the control value for any genotypes [35]. Malik et al. [36] also suggested root growth recovery as a key to waterlogging stress tolerance. Similar results were also revealed by Ahmed et al. [37] while working on mung bean under waterlogging stress.

Now let us find out why the plant tolerated the stress for the first 120 days. The answer lies in the nitrogen release time of the SCU used in the current study, which was 120 days. In the first phase, wheat could withstand all the stresses due to the soil's high accessibility of nutrients and water availability [33,34]. Our findings disclosed that crops reached maturity phases a bit early; this might be due to controlled release/coated fertilizer [20]. The experiment showed that the N released by SCU attained maximum nitrogen content in the control plants, followed by waterlogging, salt stress, combined, and then heat stress. Our results agreed with Praharaj et al. [38] and Joshi et al. [39], who worked on pulses and rice, respectively. These studies found that adequate irrigation and controlled nitrogen supplies could induce extra productivity in plants in abiotic stress conditions. The coated urea also helped the wheat plant attain better grain weight (1000-grain weight) than the control (Table 1), but fertilizer was the least effective in heat stress. Similar results of enhanced grain weight in the wheat plant were observed by Ghafoor et al. [22] while applying SCU to alleviate stress on the plant in an arid climate.

In our experiment, heat stress significantly (*p* ≤ 0.05) reduced plant growth among all stresses (Figures 2–6). This can be explained in terms of the heat sensitivity of the plant [8]. High-temperature stress is the leading environmental factor that limits wheat yield. Wheat yield decreases by 10% for every 1 ◦C increase above the mean temperature of 23 ◦C [6]. High-temperature stress affects more than 40% of the world's wheat area every year. It reduces wheat yield through chronic stress resulting from prolonged, relatively high temperatures up to 32 ◦C, or through heat-shock caused by abruptly but comparatively brief exposure to 33 ◦C and above. High temperatures cause changes in the physiological, biochemical, and molecular components of wheat crops. The high temperature might have initially accelerated the thylakoid membrane breakdown, resulting in electrolyte leakage and disruption of all electrochemical processes, particularly photosystem II (PS II) and cytochrome f/b6-mediated reactions have resulted in a drastic decrease in the photosynthesis rate [40,41]. Wheat's PS II is more exposed to extreme temperature stress, as it is a winter season crop instead of a warm-season crop, such as rice and pearl millet (*Pennisetum glaucum*) [42].

One argument can be further made based on photophosphorylation; high-temperature stress also tends to cause a halt in photophosphorylation due to thylakoid membrane damage [43]. The rate of photosynthetic CO2 assimilation was lower after stress application than in control (Figure 7b). This has previously been reported in various plant studies. The decrease in net photosynthesis could be attributed to changes in leaf water potential (Figure 7c), stomatal conductance (Figure 7b), the amount or activity of photosynthetic enzymes, and chlorophyll (Figure 4). According to some experiments, one possible factor in reducing photosynthesis in plants grown under heat stress is the accumulation of carbohydrates in leaves, indicating a feedback inhibition of photosynthesis [43,44]. Other studies have suggested that abiotic stress, particularly salt stress, reduces net respiratory activity in the roots, asserting a feedback mechanism that uses photosynthesis and inhibits plant growth [45,46]. Based on our findings, it appeared that stomatal closure may have contributed to the decreased photosynthetic rate in this experiment, particularly once the nitrogen source, i.e., SCU, was exhausted. Our results are supported by past literature, in which it was found that the nitrogen release timing of the fertilizer was key to the stress survival and nitrogen release from the slow-release fertilizer help in the recovery of the wheat plant once exposed to heat [8], salt stress [11], and waterlogging stress [17].

The wheat plant was able to grow under stress for the first 120 days. The controlled release of nitrogen fertilizer was key to the wheat plant growth, especially in waterlogged and salt stress. Many studies found that a higher grain yield can be attained by applying controlled-release SCU fertilization [23]. SCU increased grain yield efficiently by lowering rhizosphere pH, and the results were consistent with findings of a previous study [47]. Our findings also showed that using SCU N at a rate of 130 kg ha−<sup>1</sup> resulted in better growth in the first 120 days in terms of leaf area, biomass gain, photosynthesis rate, fluorescence, SPAD value, etc. However, grain yield, number of grains per spike, grain weight, and harvest index were compromised. These results are supported by past studies that reported the effects of abiotic stress on cardoon [11], wheat genotype [32], and other cash crops [35].

The earlier phase of the experiment (120 days) revealed that controlled-release fertilizers increased total N percent with equal N level application vs. later stages once the nitrogen source, i.e., SCU, was exploited [22]. After stress application, grain yield was decreased by 9.58 to 11.21%, and N uptake was reduced by 19.06 to 23.94 % (Table 4). This was because protein contains nitrogen as an essential constituent, and N is involved in all vital processes of plants. For this reason, nitrogen application is both necessary and unavoidable for crop production [20]. The optimal soil N content increases photosynthetic processes, leaf area production, leaf area duration, and net assimilation rate [33,37]. Since crop yields have increased globally due to increased N use and good management practices [19], all plants, including cereals, oilseeds, fiber, and sugar-producing plants, require a balanced amount of nitrogen for vigorous growth and development in a larger harvest with higher quality. Nitrogen fertilization has also improved Pakistani crops' growth and yield parameters for crops such as wheat, rice, sugarcane, and cotton. Wheat growth and yield parameters such as plant height (cm), number of tillers (m−2), number of spikelets (spike−1), grains (spike−1), and 1000-grain weight have been improved by nitrogen fertilization. Ali et al. [48] also demonstrated that coated urea fertilizer with higher nitrate contents and neem nitrification increased grain yield. Hence, SCU is effective under abiotic stresses once it can control nitrogen release but is no longer effective once the limit is reached. It is suggested to use a more advanced fertilizer with a better nitrogen release period of about 160 days.

Furthermore, screening should be done in soil rather than potting mix. The adverse effects in Vertosol soil were much more apparent and more representative of the actual situation on farms. The chlorophyll fluorescence model proved to be the most suitable for large-scale programs when choosing wheat genotypes for abiotic stress tolerance, requiring only a few seconds per sample. More specific studies at the cellular and tissue levels are needed to understand the fundamental physiological mechanisms fully.
