Ethylene Chemotaxis and Effectiveness of Selected Plant Growth-Promoting Rhizobacteria (PGPR) in Plant Growth Promotion under Salinity and Drougt Stress

Abstract

With the growing emphasis on sustainable agriculture, food security, and environmental protection, the use of beneficial soil microbes is imperative. Abiotic stresses, including salt and drought stress restrain the crop yield. Moreover, persistent climate changes also strengthen the frequency, degree, and damages of plants under stressed circumstances. Despite the evolution of complex abiotic stress mechanisms in modern plants, beneficial soil bacteria are also becoming more effective for mitigating these stresses. Induced stress tolerance by these soil bacteria can contribute in improvement of several soil properties by releasing extracellular compounds aimed to function as chemical signals known as chemotactic responses. Furthermore, these soil bacteria, particularly P. fluorescens can sense and swim towards ethylene (ethylene chemotaxis) and thus modulates the ethylene levels, which ultimately lowers the impact of a range of different stresses on plants. Current study consists of three main parts, the first part aimed to evaluate the chemotactic responses of various Pseudomonas species strains, the second part describes the role of two selected Pseudomonas strains (P. flourescens SBW25 and P. putida KT2440) in stress tolerance against salinity and drought stresses in barley plants. Third part describes the cloning and expression analysis of potential ethylene receptor genes for future studies to explore different pathways in response to ethylene chemotactic responses and stress tolerance. Colonization of plant roots by inoculated bacteria is an important step carried out through bacterial motility and chemotaxis. In the first part of our study, we investigated the chemotactic responses of some Pseudomonas species strains using ethylene chemotaxis assay. Our findings revealed that, except P. fluorescens F113 which showed a light zone, all the strains (Pfo-1, Pf-5, SBW25, PAO-1, PA14 and KT2440) of selected PGPR species displayed strong chemotactic responses by making dense zones around ethylene and amino acids (lysine or casamino acid) agarose plugs. The positive chemotactic responses of most of the Pseudomonas strains towards ethylene indicate their effectiveness in overcoming the environmental stresses and enhancing the plant stress tolerance. In the second part of study, two Pseudomonas species strains (P. fluorescens Abstract Ethylene Chemotaxis and Effectiveness of Selected Plant Growth-Promoting Rhizobacteria (PGPR) in Plant Growth Promotion under Salinity and Drought Stress 2 SBW25 and P. putida KT2440) were investigated for their use in plant tolerance under stressed conditions, both in soil and hydroponic systems. Barley (Hordeum vulgare L.) plants inoculated with selected PGPR strains were subjected individually to 200mM and 1000mM salt stress as well as drought stress conditions and were analyzed at the physiological, molecular (qRT-PCR) and elemental levels in comparison to non treated plants. Analysis of physiological parameters revealed that inoculated plants showed a higher germination rate, root and shoot biomasses, water potential, and chlorophyll content in soil and Hoagland solution. Similarly, higher soluble protein and osmolytes (proline, soluble sugar and free amino acids) accumulation, and lower lipid peroxidation were also observed in treated plants as compared to control plants under stressed conditions in the soil system. Similarly, our data also depicted that PGPR application led to a significant increase in the activities of some of the antioxidant enzymes (SOD, POD, CAT, and APX) under salinity stress conditions. On the other hand, under drought a significant decrease in CAT activity was observed with both PGPR strains and of Apx with P. flourescens SBW25 strain application. Root architectural analysis of 200mM salt-stressed barley roots grown in Hoagland solution showed a significant increase in root length with PGPR application. A significant increase in the length of PGPR treated barley roots was also observed under salt and drought stress conditions in the soil system. Similarly, other root parameters such as average root width (diameter), network depth and bushiness, network area, and surface area showed improvement under stress condition in PGPR applied barley roots in Hoagland solution. Further, investigation of various phytohormones (ABA, JA, ethylene, SA and IAA), stress responsive transporters and antioxidant enzymes genes (in the soil system) at molecular level revealed that, most of the JA biosynthesis pathway genes (FAD3, LOX1, AOS, AOC, OPR3, PLDα1and PI (SD10)) in either one or both PGPR applied roots were up-regulated under saline (200mM and 1000mM) conditions; while under drought stress FAD3, LOX1, LOX2, AOS, AOC, and OPR3 genes depicted a reduction and PLDα1 and PI (SD10)) genes depicted induction in PGPR inoculated plants with respect to non-treated stressed counterparts. Similarly, with PGPR applications, ethylene biosynthesis ACCS and ACCO genes displayed up regulation in salt (200mM and 1000mM) stress and down-regulation in drought stress Abstract Ethylene Chemotaxis and Effectiveness of Selected Plant Growth-Promoting Rhizobacteria (PGPR) in Plant Growth Promotion under Salinity and Drought Stress 3 conditions in comparison to non-inoculated plants under the same stress conditions. PGPR strains also helped in significant reduction of ABA biosynthesis NCED gene expression, while showed induction (with either one or both strains) of proline accumulator P5CS1 gene under salt and drought stress conditions with respect to their non-inoculated counterparts. ABA regulated genes were also modulated in PGPR treated barley roots. When treated with PGPR, DHN1 showed higher expression level with respect to non-treated plants under salt (200mM and 1000mM) and drought stress conditions, while expression of DHN5 gene was boosted in 1000mM salt stress upon PGPR applications with respect to non-inoculated stressed plants. PGPR inoculations helped in coping with the salinity (200mM and 1000mM) and drought stress conditions by significant repression of HvDRF1 gene expression with respect to non-inoculated stressed counterparts. Similarly, under the same stressed conditions, a decrease in WRKY18 expression level was also recorded in treated plants compared to control (non-inoculated) plants. In the case of SA biosynthesis pathway, ICS gene was significantly induced with P. putida KT2440 application in 200mM salt stress; while a significant reduction of this gene was noticed in 1000mM salt-stressed and drought-stressed PGPR treated roots when compared to non-treated stressed roots. MAPKK gene displayed significant induction with P. putida KT2440 inoculation in saline (200mM and 1000mM) conditions, while a reduction of this gene was observed with PGPR applications in drought stress condition with respect to non-inoculated drought stressed plants. In the case of IAA biosynthesis pathway, transcript levels of TDC, T5M and amidase genes were significantly down-regulated in PGPR inoculated plants with respect to non-treated plants under drought stress condition. On the other hand, under saline conditions, TDC and T5M genes depicted induction, while the Amidase gene showed significant reduction with PGPR (either one or both strains) applications when compared to non-inoculated barley roots under the same stress conditions. While, one of the PGPR strain (P. putida KT2440) displayed significant induction of NHX1 antiporter gene with respect to non-treated roots in 1000mM salt stress and drought stress conditions. Similarly, in comparison to non-treated plants, nitrate transporter NRT2.2 gene was significantly induced in P. fluorescens SBW25 treated plants under 200mM salt stress and drought stress conditions. In the case of Abstract Ethylene Chemotaxis and Effectiveness of Selected Plant Growth-Promoting Rhizobacteria (PGPR) in Plant Growth Promotion under Salinity and Drought Stress 4 antioxidant genes, P. fluorescens SBW25 treatment displayed significant up regulation of CAT2 gene under saline (200mM and 1000mM) conditions, while a down-regulation of this gene was noticed in PGPR inoculated drought-stressed barley roots with respect to their non-inoculated counterparts. PGPR treatments also enhanced the expression level of GR antioxidant gene in stressed (salinity and drought) conditions, with significant induction observed with P. putida KT2440 strain application when compared to non-treated plants under the same stressed conditions. Further, Particle induced X-ray emission (PIXE) technique was used for the elemental analysis of PGPR inoculated and non-inoculated plants (in the soil system) under stress vs. no stress conditions. Our PIXE analysis of various macro and micronutrients revealed enhancement of Ca, Mg, K, P, S, Al, and Si uptake in PGPR treated plants. PGPR applications depicted reduced Cl contents in 200mM salt stressed barley roots and stems, while displayed a significant increase in the barley leaves under the same stress condition. In 1000mM salt stress, a reduction in the Cl contents was observed in PGPR applied barley roots, stems, and leaves. On the other hand, a significant increase in the Cl contents was noticed in PGPR applied barley roots, stems, and leaves under drought stress condition. PGPR application also found to be effective for enhancing the uptake of micronutrients (Mn, Fe, Co, Ni, Cu, and Zn) in barley plant parts under control normal growth conditions and also under stressed conditions. Overall, these findings suggest the effectiveness of these Pseudomonas strains in improving the productivity of barley plants by reducing the adverse effects of salinity and drought stresses. In the third part of our study, bioinformatics analysis revealed putative ethylene receptor-like gene in various P. fluorescens strains including one of our selected P. fluorescens F113 strain; while during the bioinformatics analysis of barley a natural mutation was also observed at position 65 where Cysteine has been replaced with Glycine in one of its ethylene receptor ETR4. So, in order to know the role of putative ethylene receptors like gene in ethylene sensing and binding and also to know about the effect of barley ETR4 natural mutation on ethylene binding, these genes along with positive (i.e. Pseudomonas aruginosa PAO-1, MCP (tlpQ) gene, Arabidopsis thaliana ethylene receptor gene ETR1, and barley ethylene receptor genes ERS2, ETR2) and negative (pTXB1 vector) controls were successfully cloned Abstract Ethylene Chemotaxis and Effectiveness of Selected Plant Growth-Promoting Rhizobacteria (PGPR) in Plant Growth Promotion under Salinity and Drought Stress 5 and expressed in bacterial and yeast expression strains for future ethylene binding assay. Overall, our findings revealed favorable chemotactic behavior of PGPR and PGPR based improvement of physiological parameters and modulation of major defense mediated pathways, ion transporter, nitrate transporter, and antioxidant enzymes, as well as an improvement in the uptake of macro and micronutrients for the enhancement of salinity and drought tolerance. Both PGPR strains showed improved effects on the parameters under stressed conditions. Conclusively, these PGPR species are an effective source of plant stress tolerance and elevated growth of barley plants. Published Research Paper from This Study: Modulation of Barley (Hordeum vulgare) defense and hormonal pathways by Pseudomonas species accounted for salinity tolerance. Sania Zaib1,*,# , Imtiaz Ahmad1, 2, # and Samina N. Shakeel1,*. Pak. J. Agri. Sci. Vol. 57(6), 1469-1481; 2020