Deciphering salt tolerance mechanisms in synthetic hexaploid and bread wheat under humic acid application: physiological and genetic perspectives

Abstract

Salt stress is a potential constraint that perturbs plant physiological and osmolytic processes, and induces oxidative stress. The plant biostimulant, such as humic acid (HA) is capable to improve the wheat-tolerance to salt stress through triggering the plant defense mechanisms and regulating the genetic determinants. In this context the present study has comparatively evaluated the effect of HA on salt tolerant synthetic hexaploid (SH) and salt susceptible bread wheat (BW) genotypes. The experiment was performed in three replicates using randomized complete block design (RCBD) having two factorial arrangements, with HA treatment as one, while genotype as second factor. HA treatment significantly enhanced chlorophyll (33.33%-100%) and photosynthesis (31.25%-50%), and significantly reduced the glycine betaine (GB) (42.85%-77.77%), proline (20%-28.57%) and Na⁺/K⁺ ratio (33.33%-50%) in salt stressed SH and BW genotypes. Additionally, HA significantly increase the activities superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) by 57.14%-66.67%, 54.54%-83.33%, and 55.55%-80%, respectively in all salt stressed genotypes. The salinity associated genes TaNHX1, TaHKT1,4, TaAKT1, TaPRX2A TaSOD and TaCAT1 were upregulated, while TaP5CS was downregulated in SH and BW genotypes corresponding to their regulatory traits. Furthermore, the multivariate analysis including correlation, principal component analysis (PCA) and heatmap dendrogram further rectified the strong impact of HA on the strength of association and expression of stress marker traits. Overall, the SH genotypes showed more strong response to the HA and illustrated significant tolerance to salt stress based upon physiological, biochemical and genetic indicators. Conclusively, the SH can serve as a bridge to transfer alien genes associated with salt tolerance into elite bread wheat germplasm.

Keywords: antioxidant; correlation; gene regulation; heatmap; synthetic hexaploid.

Figure 1

Effect of HA and control treatment on chlorophyll, photosynthesis, proline and glycine betaine content of different wheat genotypes grown under salt stressed environment. Chl, chlorophyll; Pn, photosynthesis; GB, glycine betaine. Graph bars represent the mean values of traits, analysed during tri-replicated two factorial experiment at p ≤ 0.05. The bar values following the different letters are significantly different at p ≤ 0.05.

Figure 2

Effect of HA and control treatment on the activities of antioxidant enzymes (SOD, POD, and CAT) and Na⁺/K⁺ ratio of different wheat genotypes grown under salt stressed environment. SOD, superoxide dismutase; POD, peroxidase; CAT, catalase. Graph bars represent the mean values of traits, analysed during tri-replicated two factorial experiment at p ≤ 0.05. The bar values following the different letters are significantly different at p ≤ 0.05.

Figure 3

Correlogram showing the effect of control and HA treatment on the paired association of chlorophyll, photosynthesis, osmolytes (Proline and GB), Na⁺/K⁺ and antioxidant enzymes (SOD, POD and CAT) in salt stressed wheat genotypes. Chl, chlorophyll; Pn, photosynthesis; GB, glycine betaine; SOD, superoxide dismutase; POD, peroxidase; CAT, catalase. ***Significant at p ≤ 0.001, **Significant at p ≤ 0.01.

Figure 4

The PCA biplot based upon correlation matrix, indicating the effect of humic HA and control treatment on chlorophyll, photosynthesis, osmolytes (Proline and GB), Na⁺/K⁺ and antioxidant enzymes (SOD, POD and CAT) in salt stressed wheat genotypes. The orientations and closeness of traits vectors differs under HA application as compared to control treatment which confirms the changing association and expression of traits with respect to genotypes categorized into different biplot quadrants. Chl, chlorophyll; Pn, photosynthesis; GB, glycine betaine; SOD, superoxide dismutase; POD, peroxidase; CAT, catalase.

Figure 5

The varying dispersion of ellipses in biplot indicates the strong impact of HA treatment on chlorophyll, photosynthesis and antioxidant enzymes (SOD, POD and CAT), and strong impact of control treatment on osmolytes (Proline, andGB) and Na⁺/K⁺ in salt stressed wheat genotypes. Chl, chlorophyll; Pn, photosynthesis; GB, glycine betaine; SOD, superoxide dismutase; POD, peroxidase; CAT, catalase.

Figure 6

The varying dispersion of ellipses representing salt tolerant SHs (SH1-SH4) and salt susceptible BW in biplot, indicates the high expression of chlorophyll, photosynthesis and antioxidant enzymes (SOD, POD and CAT) in SH, and high expression of osmolytes (Proline, GB) and Na/K in BW genotypes. Chl, chlorophyll; Pn, photosynthesis; GB, glycine betaine; SOD, superoxide dismutase; POD, peroxidase; CAT, catalase.

Figure 7

Heatmap categorizing the salt stressed wheat genotypes in terms of the varied trait expression under humic acid and control treatment. The varying colour pattern of bands from light to dark represent the change in the expression of trait from low to high due to the application of HA and control treatment. Chl, chlorophyll; Pn, photosynthesis; GB, glycine betaine; SOD, superoxide dismutase; POD, peroxidase; CAT, catalase.

Figure 8

Relative expression analysis of salt stress related genes in susceptible BW and SH wheat genotypes under the application of HA and control treatment.

Figure 9

Schematic representation indicating the impact of HA on physiological, biochemical and molecular mechanisms imparting salt stress tolerance. Chl, chlorophyll; Pn, photosynthesis; GB, glycine betaine; SOD, superoxide dismutase; POD, peroxidase; CAT, catalase.