Investigating the molecular mechanism and genetic utility of plant growth-promoting rhizobacteria-mediated induced systemic tolerance

Abstract

Drought is a major abiotic stress limiting agriculture production. Most research is focused on identifying and subsequently overexpressing plant drought-responsive genes (DRGs) to enhance the drought tolerance of plants. However, such research has shown undesired phenotypes on plants as a side effect. Recently, two DRGs (RD)29A and RD29B were identified to be induced by Paenibacillus polymyxa CR1, a plant growth-promoting rhizobacterium capable of priming drought tolerance (i.e., Induced Systemic Drought Tolerance, IST) and concurrently stimulating plant growth, play pivotal roles in defense responses against drought. This study was conducted in order to replicate the IST molecular mechanism of PGPR P. polymyxa CR1 by overexpressing RD29 genes, aimed to circumvent the growth trade-off typically associated with drought tolerance in plants using strong circadian COR15A promoter. Compared to strong constitutive 35S promoter, native RD29 circadian promoters, strong circadian COR15A promoter showed normal growth in optimal conditions. These results hold promise for generating economically viable drought-tolerant crops. Similarly, during drought stresses, antioxidant enzymes such as Glutaredoxin (GRXs) are produced to maintain the cellular redox homeostasis in plants and generally divided in 3 classes in Arabidopsis model plants. These are a heat-stable oxidoreductases that use reduced glutathione (GSH) to metabolize thiol-disulfide exchanges with protein-disulfides, namely ‘deglutathionylation’. Specially, the class III GRXs functions in various hormone signaling to coordinate and survival under environmental stresses. For instance, GRX480 is induced by jasmonates and salicylic acid, and in turn binds/controls TGA transcription factors (TFs), which actuates general defense mechanisms. However, the biochemical activity and mode of GRX480 have not been characterized due to its C-terminus hydrophobic tail that hinders the expression and purification of recombinant proteins. In 2 this study, we employed a maltose-binding protein (MBP) as a solubility enhancer, and successfully prepared a recombinant GRX480. Using the plasmid pKDL66, an amino (N)- terminus HIS-tagged MBP was fused to the N-terminus of GRX480 and induced under the control of T7 promoter in Escherichia coli BL21 (DE3) cells. The HIS-MBP tag was later removed by tobacco etch virus proteases, and the nickel affinity column chromatography. GRX480 confirmed its oxidoreductase activity, reducing 0.39 μM of S- glutathionylted bovine serum albumin per min ratio. Its apparent Km values were determined as 6.4 μM, lower than known Km values of class I GRXs (> 80 μM), suggesting that increased GSH levels induced by jasmonate and salicylic acid signaling could re- arrange GRX480 and TGA interactions, and TGA-dependent transcriptions.