Drought stress hampers the chlorophyll contents and carotenoids
Drought stress and cultivars significantly (P < 0.05) affected the production of photosynthetic pigments, i.e., Chl a, Chl b, total chlorophyll (Chl a + b), and carotenoids, whereas no interaction was found statistically significant (P < 0.05). The photosynthetic pigments decline rates were higher with an increase in drought stress with the highest rate at severe water deficit in all Axonopus compressus cultivar (Fig. 2). The values of percentage reduction in Chl a, Chl b, and total chlorophyll content (Chl a + b) were recorded as 8–27, 34–77, and 17–43%in A-58, 12–25, 48–74, and 22–39% in A-59 and 19–33, 44–73, and 25–43% in A-38, as compared with control. A-58 gave significantly higher values 1.423, 0.667 and 2.092 of the Chl a, b and a + b respectively, while minimum values were found in the A-38. Moreover, the change in chlorophyll contents due to drought was more severe at higher levels of drought in all Axonopus compressus accessions. Overall, the A-58 performed better in terms of the chlorophyll a, b and a + b contents than A-59 and A-38. While the magnitude of drought stress on photosynthetic pigments across cultivars was recorded as followed A-58 > A-59 > A-38 (Fig. 2a-d).
Drought stress inhibited growth and leaf water status of Axonopus compressus.
Significant effects were observed in morphological traits and leaf water potential due to Drought stress in Axonopus compressus, whereas no interaction effect was found significant except in case of leaf length and area. The highest values 46.13, 4.30, 0.97, and 4.71 cm of morphological traits i.e; stem length, leaf length, leaf width, and leaf area respectively, were observed in A-58. Moreover, greater value of leaf water potential was also observed in A-58 (Table 2). The highest decrease 36, 11, 32, and 15% of stem length, leaf length, leaf width, and leaf area respectively due to drought induction was observed in A-38 (Table 2). The reductions in morphological traits were increased with an increase in drought levels from low to high drought in all accessions. Overall, the result showed A-58 proved tolerant than other two accessions (A-38 and A-59) and drought-induced damage was more intense in A-38 than those in A-59 or A-58.
Drought stress-triggered oxidative damage and osmolyte accumulation.
Drought-induced oxidative stress in terms of enhanced H2O2 production, lipid peroxidation, and membrane damage; though levels were fairly higher at severe drought for all A-58, A-59 and A-38 (Fig. 3). The production of H2O2 was 33.09-100.88, 32.90-93.06 and 40.61-125.05% in A-58, A-59 and A29 respectively, Similarly, electrolyte leakage and MDA production were enhanced linearly with increasing drought level, maximum at severe drought level i.e., 21.03 and 110.26% (for A-58), 33.55 and 123.26% (for A-59), 37.72 and 149.59% (for A-38). Drought stress affected TBARS accumulation significantly (P < 0.05) but the cultivars were remained statistically similar (P > 0.05). Overall, the rate of oxidative stress was higher at high drought stress and was more prominent in A-38 than A-58 and A-59.
Both drought stress and accessions affected total phenols and proline accumulation significantly (P < 0.05) whereas, no interaction was found statistically significant (P < 0.05) (Fig. 4a,c). While in case of the soluble sugars and protein the interactions were found statistically significantly (P > 0.05). Higher values 40.03 and 67.31, 38.84 and 62.53, 31.09 and 68.08 of proline and total phenols were estimated in the A-58, A-59, and A-38 respectively,under severe drought. In addition, both soluble sugar and soluble proteins accumulation were increased with an increase in drought induction in all accessions. Furthermore, increase of soluble sugar and soluble proteins accumulation were ranged from 15.95–41.29 and 17.13–78.59%, 3.57–38.11 and 11.64–60.59%, 13.54–22.84 and 17.46–54.67% in A-58, A-59 and A-38 respectively (Fig. 4b, d).
Drought-induced regulations of enzymatic and non-enzymatic antioxidant activity.
Drought stress and accessions significantly (P < 0.05) affected enzymatic (SOD, POD, CAT, and APX) and non-enzymatic (GSH, GSSG, total glutathione contents (GSH + GSSG) whereas, GSH/GSSG levels were found non-significant regarding stimulation of the antioxidants. Furthermore, the drought × accession interaction was extended up to significant level (P < 0.05) for SOD, CAT, POD, APX, GSSH and total glutathione contents (GSH + GSSG).
For A-58 the activities of CAT, POD, SOD, and APX increased by 7.23, 42.67, 30.32, and 27.57% under severe drought, as compared with control. Whereas, the activities of the enzymes were changed by -8.31 and − 35.13, 33.57 and − 19.58, 30.19 and − 4.29, 19.56 and − 47.51% for the A-59 and A-38 in severe drought (Fig. 5a-d). Antioxidant activities were found higher at high drought level while decreased abruptly as drought level increased form medium to high, especially in A-38. Furthermore, activity of SOD increased 7.85 and 10.20% till medium to high drought level (for A-58 and A-59 respectively), while the activity decreased 7.90% in A-38 for medium to high drought level. POD activity increased with increase in drought severity (maximum at medium drought level i.e., 44.49 and 39.34% in A-58 and A-59, respectively), CAT activity enhanced by 56.15% up to medium drought-level then decreased with an increase in drought level (in A-58) and for A-59 highest (41.77%) activity at medium drought level. The activities of APX increased with an increase in drought-levels in both A-58 and A-59 with highest values of 27.57 and 19.56% respectively, while, in A-38, the APX activity decreased with increase in drought stress with highest value (13.14%) at low drought level. Overall, antioxidant enzymatic activities were found higher in A-58 than both A-59 and A-38 (Fig. 5).
Furthermore, drought significantly changed (P < 0.05) GSH, GSSG and total glutathione (GSH + GSSG) in the Axonopus compressus leaves. For A-58, plants exposed to severe drought level accumulated highest (18.31%) GSH contents, similar trends were observed for GSSG (9.09 and 7.23%) and total glutathione (GSH + GSSG) (10.71 and 8.66%) contents in both A-59 and A-38. Nonetheless, GSH/GSSG ratio was found non-significant in all accessions (Fig. 6a-d).
Effect of Drought Stress on the expression level of the drought-responsive genes and transcription factors in A. Compressus
TFs determine the expression of genes in plants. When drought and high-temperature stress disorders happen, the plant TFs expression promotes to alter the expression of downstream responsive genes that improve the ability to resist stress. The stress responses in plants are strongly linked to TFs i.e., MYB, WRKY, NAC, and DREB; that support plants to normalize their functioning in unfavorable drought conditions. Drought stress influenced the TFs expression in A. compressus during the experiment (Fig. 7).
In the current study, the MYB and WRKY1 gene average expression levels under drought stress were 2.89 fold and 1.9 fold higher in A-58 than those in the control A. compressus plants respectively. While for the DREB and NAC genes the 1642.83 fold and 39.96-fold higher values were observed in A-58 during drought treatment than well water control. The expression levels of drought-responsive genes (PIP1, ABI5, and MAPK1) were higher under drought stress treatments when compared to those in the untreated control A. compressus (Fig. 7A, B, C). The PIP1 and ABI5 show elevated expression with values of 5.96 fold and 12.98 fold higher than well water control (Fig. 7E, F). Similarly, the MAPK also showed significant expression level value of 5.43 folds than well water control (Fig. 7G).