Unraveling Morphophysiological and Biochemical Responses of Triticum aestivum L. to Extreme pH: Coordinated Actions of Antioxidant Defense and Glyoxalase Systems

Soil pH, either low (acidity) or high (alkalinity), is one of the major constraints that affect many biochemical and biological processes within the cell. The present study was carried out to understand the oxidative damage and antioxidant defense in wheat (Triticum aestivum L. cv. BARI Gom-25) grown under different pH regimes. Eight-day-old seedlings were exposed to growing media with different pH levels (4.0, 5.5, 7.0, and 8.5). Seedlings grown in pH 4.0 and in pH 8.5 showed reductions in biomass, water, and chlorophyll contents; whereas plants grown at pH 7.0 (neutral) exhibited a better performance. Extremely acidic (pH 4.0) and/or strongly alkaline (pH 8.5)-stress also increased oxidative damage in wheat by excess reactive oxygen species (ROS) generation and methylglyoxal (MG) production, which increased lipid peroxidation and disrupted the redox state. In contrary, the lowest oxidative damage was observed at a neutral condition, followed by a strong acidic condition (pH 5.5), which was mainly attributed to the better performance of the antioxidant defense and glyoxalase systems. Interestingly, seedlings grown at pH 5.5 showed a significant increase in morphophysiological attributes compared with extreme acidic (pH 4.0)- and strong alkaline (pH 8.5)-stress treatments, which indicates the tolerance of wheat to the acidic condition.


Relative water and proline content
The contents of Pro were boosted up upon exposure to extreme pH condition in wheat 125 seedlings, and compared to control 19-and 4-fold increase in Pro content was found in acid-stressed 126 seedlings (pH 4.0 and pH 5.5, respectively). Alkaline-stress (pH 8.5) also gave rise to Pro content by 127 22-fold compared to control (Table 1). increased by 199 and 194% in pH 4.0 and pH 8.5 exposed seedlings respectively. However, seedlings 132 grown on pH 5.5 showed lower increases in MDA content (95%) compared with control seedlings.

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In line with MDA, a remarkable raise in H2O2 content was noticed in leaf tissue upon exposure 134 to varying rhizosphere pH (Figure 2b). Compared with control a sharp increase in H2O2 content (134, 135 and 90%) was observed both at extremely acidic (pH 4.0) and strongly alkaline (pH 8.5) pH.

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Similarly varying rhizosphere pH increased the activity of lipoxygenase (LOX) drastically.

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Upon exposure to extreme pH, LOX activity increased by 73 and 67% (compared with control) in extremely acidic (pH 4.0) and strongly alkaline (pH 8.5) stressed seedlings, respectively. But  Extreme rhizosphere pH destroyed chl in leaf tissues. Chlorophyll a content was decreased in acid-stressed wheat seedlings in a dose-dependent manner by 13 and 8% at pH 4.0 and pH 5.5, to control 31 and 15% decrease in chl b content was found in acidic condition pH 4.0 and pH 5.5 153 respectively, while compared to control 28% reduction in chl b content was found in alkaline (pH 8.5) condition ( Figure 3b). Extreme pH-stress once more reduced chl (a+b) content; in comparison 155 with the control seedlings. Hence, the chl (a+b) values were decreased by 19 and 10% compared with 156 control in acid-stress, pH 4.0 and pH 5.5 treated seedlings, respectively (Figure 3c), whereas extreme pH, and compared to control 30 and 16% reduction was observed in acidic (pH 4.0 and pH 160 5.5 respectively) condition, while 28% reduction in car content was found at in alkaline (pH 8.5) 161 condition ( Figure 3d). The chlorotic symptoms were also visible in the leaves in seedlings exposed to  the decrease in GSH content occurred upon exposure to extreme acidic condition, whereas 15% decrease in GSH content was found in strong alkaline-stress (pH 8.5) compared with the control increased GSSG content by 106% compared control (Figure 4e). The ratio of GSH and GSSG was also 185 changed due to extreme pH-stress. In response to acidity-stress, 52 and 25% decrease was observed 186 in GSH/GSSG ratio under pH 4.0 and pH 5.5, respectively. Whereas, 58% decrease in GSH/GSSG 187 ratio was observed in strongly alkaline (pH 8.5)-stress ( Figure 4f). 228 reduction in Gly I activity was attributed to alkaline (pH 8.5) growing condition compared to 229 control. Meanwhile, in respect to control, Gly II activity was reduced by 12, 5 and 27% in extreme pH 230 condition (pH 4.0, pH 5.5 and pH 8.5, respectively) of the growing media (Figure 7b).

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Because of down-regulation of these two vital enzymes, MG content increased notably.

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Compared with the control seedlings MG content was increased by 35 and 8% in acidity-stressed 233 (pH 4.0 and pH 5.5) seedlings, while a sharp increase (78%) in MG content was observed when the 234 seedlings were exposed to strong alkaline condition (Figure 7c).

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Hence both acidity and alkalinity increase the risk of drought stress.

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In the present study, we found the negative effects of extreme pH both acidity-and  276 found low pH (2.5) induced alteration of chl pigments contents, which further reduce the 277 photosynthetic capacity as well as CO2 assimilation in Citrus. But they didn't find any changes 278 regarding the photosynthetic efficiency at pH ≥2.5. However, the differential response to same stress 279 may happen due to the genetic makeup, which indicate more tolerance of citrus to acidity stress.

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We found reduced RWC in leaf tissue of wheat seedlings due to extreme pH, both acidity and 281 alkalinity, which might be attributed to the reduce root length due to alteration in growing media 282 pH, which subsequently caused water unavailability in the growing shoot, and induces artificial extreme acidity stress, hence the enzyme molecules might be unable to release their product due to under extreme pH-stress, the H2O2 content was not reduced, hence it can be mentioned that the upregulation of CAT activity was not enough to scavenge the overproduced H2O2, which indicates systems activated to detoxify the overgenerated ROS and MG, but fails after a certain extent.
(pH 5.5) performed better compared with the seedlings grown under extreme acidic (pH 4.0) and dirt, debris, small sized or dead seeds. Separated quality seeds were then surface sterilized with 1%

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(total and reduced) was assayed at 265 nm spectrophotometrically using a standard curve and DHA 434 was calculated after subtracting reduced AsA from total AsA [14]. Glutathione was determined by 435 enzymatic recycling and the rate of absorption change was read by spectrophotometer at 412 nm and 436 plotted against standard curves with known concentrations of GSH and GSSG. Yet, 2-vinylpyridine 437 dependent removal of GSH was used to determine GSSG content. Finally, the content of GSH was 438 calculated by subtracting GSSG from total GSH [14].