*3.1. Biochemical Content Analysis of Primed Plants in Response to Fungal Stress* 3.1.1. Proline

The seed priming exhibited a positive effect, by stimulating all the biochemical parameters under both the priming as well as the systemic inoculation conditions. Proline performs its function as a beneficial solute under normal conditions, and as stress tolerance in non-healthy conditions [52]. In our experiment, the total proline content was significantly increased at the seedling stage after each priming treatment, as compared to the control: this significant increase was more pronounced in osmotic and hydro-primed plants—71.56% and 70.09%, respectively—followed by halo-primed and hormonal-primed plants—57.88 and 52.44%, respectively, compared to the control. After systemic inoculation of *A. niger*, the highest increase in proline content was observed in osmotic and halopriming—51.26% and 49.50%, respectively—followed by hydropriming and hormonal priming—47.30% and 4.11%, respectively, compared to the control (Figure 1A).

#### 3.1.2. Protein Content

Production of protein in stress conditions is mainly associated with plant defense responses against fungi [53]. In each priming treatment, protein content was observed to be significantly more increased than in the control. The hydro and osmotic-primed plants had no significant differences in protein content, but when compared to the halo-primed, hormonal-primed, and control plants, a significant change was observed. However, osmotic priming, hydropriming, halopriming, and hormonal priming enhanced protein content by 31.09%, 28.05, 17.00%, and 13.25%, respectively. Moreover, in systemic inoculation of *A. niger* also, a significant increase in protein content was recorded, as compared to the control in all groups. Overall, 32.93% and 22.27% increases in the protein content were observed in osmotic priming and hydropriming, followed by hormonal priming (16.29%) and halopriming (14.00%), compared to the control (Figure 1B).

#### 3.1.3. Sugar Content

Sugar is considered a primary source of energy, which acts as a building block for providing defense-responsive material in plants [54]. Our results showed that the primed plants contained more soluble sugar in their leaves than non-primed and inoculated primed plants. In essence, the osmotic-primed and hydro-primed plants showed a significant increase in sugar content, increasing by 24.60% and 24.75%, respectively, while the haloprimed and hormonal-primed plants exhibited 12.56% and 12.87% increases in sugar contents, respectively, compared to the control plants. By contrast, the systemic inoculation of *A. niger* resulted in a significant drop in sugar content in all primed plants as compared to the control, where 14.32%, 23.07%, 23.92%, and 27.45% greater reductions in sugar content

were observed for osmotic-, halo-, hormonal-, and hydro-primed plants, respectively, than in the control (Figure 1C).

**Figure 1.** Biochemical contents of wheat under hydropriming, osmotic priming, halopriming, and hormonal priming: (**A**) proline content, (**B**) protein content, (**C**) sugar content, (**D**) chlorophyll content. The mean values with different letter(s) indicate significant differences at *p* ≤ 0.05. Vertical bars represent standard deviation of means (*n* = 3). Sys. inoculation: Systemic inoculation.

#### 3.1.4. Chlorophyll Content

The photosynthetic capacity of plants is determined by their leaf chlorophyll content and measurement [55]. The results of the present study revealed that seed priming exerted a positive effect on the chlorophyll content. The hydro-primed plants showed the highest increase in chlorophyll content, of 41.54%, followed by the osmotic-, hormonal-, and halo-primed plants, which enhanced chlorophyll content by 32.14%, 29.63%, and 25.49%, respectively, as compared to the control. Similarly, under systemic inoculation, all the primed plants showed a significant increase in chlorophyll content, as compared to the control (Figure 1D). However, osmotic priming exhibited the highest chlorophyll contents (65.45%), followed by hormonal priming (51.22%), hydropriming (60.42%), and halopriming (56.82%), compared to the control.
