Plant growth promoting rhizobacteria Dietzia natronolimnaea modulates the expression of stress responsive genes providing protection of wheat from salinity stress

Abstract

Plant growth promoting rhizobacteria (PGPR) hold promising future for sustainable agriculture. Here, we demonstrate a carotenoid producing halotolerant PGPR Dietzia natronolimnaea STR1 protecting wheat plants from salt stress by modulating the transcriptional machinery responsible for salinity tolerance in plants. The expression studies confirmed the involvement of ABA-signalling cascade, as TaABARE and TaOPR1 were upregulated in PGPR inoculated plants leading to induction of TaMYB and TaWRKY expression followed by stimulation of expression of a plethora of stress related genes. Enhanced expression of TaST, a salt stress-induced gene, associated with promoting salinity tolerance was observed in PGPR inoculated plants in comparison to uninoculated control plants. Expression of SOS pathway related genes (SOS1 and SOS4) was modulated in PGPR-applied wheat shoots and root systems. Tissue-specific responses of ion transporters TaNHX1, TaHAK, and TaHKT1, were observed in PGPR-inoculated plants. The enhanced gene expression of various antioxidant enzymes such as APX, MnSOD, CAT, POD, GPX and GR and higher proline content in PGPR-inoculated wheat plants contributed to increased tolerance to salinity stress. Overall, these results indicate that halotolerant PGPR-mediated salinity tolerance is a complex phenomenon that involves modulation of ABA-signalling, SOS pathway, ion transporters and antioxidant machinery.

Figure 1. Effect of PGPR inoculation on wheat plant growth under non-saline and saline conditions.

(a–c) Plants were grown in soil under glasshouse conditions and saline plants supplemented with 150 mM NaCl via irrigation and harvested 60 d after germination. Dry weight and plant height of wheat shoots were measured. (d–f) 12 d old wheat plants grown hydroponically in Hoagland nutrient solution. NaCl was added to the nutrient solution to obtain a final concentration of 100 mM. Dry weight and height of wheat shoots were measured after 12 d of germination. Control: without bacterial treatment; PGPR: plants inoculated with Dietzia natronolimnaea STR1 strain. Values are mean of five replicates ± standard error of means. Different letters indicate statistically significant differences between treatments (Duncan’s multiple range test P < 0.05).

Figure 2. Effect of salt stress on photosynthetic pigments, proline and MDA content of PGPR-inoculated wheat plants.

Chlorophyll a, Chlorophyll b, carotenoid, proline and MDA content was measured in leaves of 60 d old wheat plants grown in soil supplemented with 150 mM NaCl and inoculated with STR1. Control: non-inoculated wheat plants, PGPR: plants inoculated with Dietzia natronolimnaea STR1. Values are mean of five replicates ± standard error of means. Different letters indicate statistically significant differences between treatments (Duncan’s multiple range test P < 0.05).

Figure 3. Real time expression analysis of TaOPR1 and ABARE in shoot and root of PGPR-inoculated wheat plants subjected to salt stress.

The expression analysis of TaOPR1 and ABARE transcript in (a) shoot and (b) root of PGPR Dietzia natronolimnaea STR1 inoculated 12 d old wheat plants under both non-saline and saline conditions. Un-inoculated wheat plants grown in non-saline condition were used as a control. The data represented means of triplicate biological and experimental repeats; error bars represented SEM. Different letters indicate statistically significant differences between treatments (Duncan’s multiple range test P < 0.05).

Figure 4. Real time expression analysis of SOS pathway genes viz., SOS1 and SOS4 in shoot and root of PGPR-inoculated wheat plants subjected to salt stress.

The expression analysis of SOS1 and SOS4 transcript in (a) shoot and (b) root of PGPR Dietzia natronolimnaea STR1 inoculated 12 d old wheat plants under both non-saline and saline conditions. Un-inoculated wheat plants grown in non-saline condition were used as a control. The data represented means of triplicate biological and experimental repeats; error bars represented SEM. Different letters indicate statistically significant differences between treatments (Duncan’s multiple range test P < 0.05).

Figure 5. Real time expression analysis of WRKY, MYB and TaST in shoot and root of PGPR-inoculated wheat plants subjected to salt stress.

The expression analysis of WRKY10, WRKY17, MYB33 and TaST transcript in (a) shoot and (b) root of 12 d old wheat plants inoculated with PGPR Dietzia natronolimnaea STR1 under both non-saline and saline conditions. Un-inoculated wheat plants grown in non-saline condition were used as a control. The data represented means of triplicate biological and experimental repeats; error bars represented SEM. Different letters indicate statistically significant differences between treatments (Duncan’s multiple range test P < 0.05).

Figure 6. Real time expression analysis of TaHKT, TaHAK and TaNHX in shoot and root of PGPR-inoculated wheat plants subjected to salt stress.

The expression analysis of TaHKT, TaHAK and TaNHX transcript in shoot and root of PGPR Dietzia natronolimnaea STR1 inoculated 12 d old wheat plants under both non-saline and saline conditions. Un-inoculated wheat plants grown in non-saline condition were used as a control. The data represented means of triplicate biological and experimental repeats; error bars represented SEM. Different letters indicate statistically significant differences between treatments (Duncan’s multiple range test P < 0.05).

Figure 7. Real time expression analysis of POD, APX, GPX, MnSOD, GR and CAT in shoot and root of PGPR-inoculated wheat plants subjected to salt stress.

The expression analysis of POD, APX, GPX, MnSOD, GR and CAT transcript in (a) shoot and (b) root of 12 d old wheat plants inoculated with PGPR Dietzia natronolimnaea STR1 under both non-saline and saline conditions. Un-inoculated wheat plants grown in non-saline condition were used as a control. The data represented means of triplicate biological and experimental repeats; error bars represented SEM. Different letters indicate statistically significant differences between treatments (Duncan’s multiple range test P < 0.05).

Figure 8. Overview of modulation of expression of genes involved in PGPR Dietzia natronolimnaea STR1-mediated salinity tolerance in wheat plants.

In the early steps of interaction, Dietzia natronolimnaea STR1 triggered signal transduction which modulated the expression of several genes responsible for salt tolerance. Based on the results we propose that carotenoid producing D. natronolimnaea STR1 participates in salt tolerance via both ABA-mediated and SOS-mediated pathways by up-regulating the expression of ABA-signalling cascade genes (TaABARE and TaOPR1), leading to induction of TaMYB and TaWRKY expression followed by stimulation of expression of a plethora of stress related genes including TaST, a salt stress-induced gene, associated with promoting salinity tolerance. Modulation of SOS pathway related genes (SOS1 and SOS4) and tissue specific responses of ion transporters TaNHX1, TaHAK and TaHKT1, were observed in PGPR applied plants. The enhanced gene expression of various antioxidant enzymes such as APX, MnSOD, CAT, POD, GPX and GR and higher proline content in PGPR-treated wheat plants contributed to increased plant tolerance to salinity stress. The red coloured bold arrows indicate the up-regulation or down-regulation of genes.