Differential expression analysis of Calmodulin-binding (CaM) gene in Pea plants under drought stress and the effects of Plant Growth Promoting Rhizobacteria and Plant Growth Regulators

Abstract

In the proceeding climate change, drought stress has been identified as the major stress factor. To survive under such conditions, plants respond these changes by manipulating key physiological processes and modulation of expression of various Calmodulin (CaM) genes. The present study was aimed to investigate the effect of plant growth hormones, salicylic acid (SA), abscisic acid (ABA) and Rhizobium pisi (strain DSM 30132) applied singly and in combination, on pea (Pisum sativum L.) cv. Florida plants under control and drought stressed conditions. Prior to the sowing, seeds were soaked for 5h in broth culture (108 cfu/ml) of Rhizobium pisi and SA /ABA. The seeds soaked for 5h in distilled water served as control. Three weeks old (21d) seedling were subjected to drought stress by discontinuing water supply and the effects were examined at two different time points of drought i.e., 4d (TP1) and 8d (TP2) of induction of drought stress. The salient physiological parameters studied were; moisture content of rhizosphere soil, plant biomass, and relative water content (RWC), canopy temperature, stomatal index, stomatal conductance, chlorophyll content, chlorophyll fluorescence and nutrient content. The inoculation effects of Rhizobium pisi and priming of SA and ABA on the expression analysis of PsCaM1 was also evaluated. The expression level of PsCaM1 (Pisum sativum Calmodulin) gene was identified by qRT-PCR among the treatments. The actin gene was used as a reference gene. Results revealed a higher retention of soil moisture content in rhizosphere soil of abscisic acid treated plants at TP1 and TP2. Abscisic acid decreased the fresh and dry weight of plants under unstressed condition but increased the fresh weight as well as relative water content under drought stress. Rhizobium and SA ameliorated the adverse effects of drought stress more effectively than ABA alone. The Rhizobium inoculation significantly increased stomatal conductance under drought stress at TP2. Under drought stress, at TP1 all the treatments alone and in combination increased the RWC significantly over drought stressed plants. The FV/FM ratio was higher in SA treatment followed by combined treatment with SA, Rhizobium and ABA. Under drought stress, both Na and K uptake was significantly increased in Rhizobium + SA + ABA and Rhizobium inoculation. ABA, Rhizobium + SA treatments enhanced the Mg uptake under drought stress. Whereas, ABA, Rhizobium + SA + ABA increased accumulation of Ca content. Fe was significantly higher in Rhizobium and combined treatment of Rhizobium + SA + ABA. Similarly, Zn and Mn accumulation was also improved in Rhizobium treatment. Expression analysis demonstrated a significant upregulation of PsCaM1 gene under drought stress. ABA showed significantly higher (~1.5 folds ) expression under drought stress at TP2x followed by Rhizobium + ABA, Rhizobium + SA that divulged an increased expression of 0.8, 0.5 and 0.4 folds respectively at long term drought stress (TP2). PsDREB2 gene is positively induced in Rhizobium, ABA and combined treatment of Rhizobium + ABA under long term drought stress (TP2). It is deduced from the data that Rhizobium alone or in association with SA may be used to mitigate drought induced inhibition on plant growth. Rhizobium, ABA and SA treatments exhibited better growth effect on pea plants at short term drought stress. Whereas, Rhizobium assisted SA and ABA to alleviate drought induced adverse effects over long term drought. The PsCaM and PsDREB2 gene is induced under long term drought stress. It is inferred that ABA, Rhizobium and consortium of ABA, SA and Rhizobium can be ideal candidate to enhance drought tolerance in pea plants by the upregulation of PsCaM gene. Keyword: Pisum sativum, Drought stress, ABA, SA, CaM, PsCaM, PsDREB