Effect of plant-derived smoke solution on nodule formation and proteomics of chickpea (Cicer arietinum L.)

Abstract

Plant-derived smoke obtained from burning vegetation has been used in agricultural and horticultural practices since last two decades. Being a cheap and environment friendly source, smoke plays an important role in seed germination, plant growth and enhancement of crops yield. The aim of the present study was to investigate the effect of plant-derived smoke on morphological, physiological, nodulation, rhizobium growth, proteomics and gene regulation in seedlings of chickpea treated with plant-derived smoke. Seeds of chickpea (C. arietinum) were sown in pots containing 500 g sands and were kept in a growth chamber having fluorescent light at 25oC and 60% humidity. For morphological parameters the seeds were supplied without or with 1000, 2000 and 4000 ppm plant-derived smoke for 2, 4, 6 and 8 days. For nodulation, seeds of chickpea were mixed with the inoculum of rhizobium having cell suspension of 109 CFU mL-1 and were sown in pots containing sands. The numbers of nodules were recorded after 11, 18 and 25 days. The rhizobium culture was measured using spectrophotometer at 600 nm incubated for 12, 24, 48 and 96 h. For physiological, proteomics, immuno-blotting and qRT-PCR, seedlings were irrigated without or with 2000 ppm for 6 days. For the analysis of proteins, gel-free/label-free proteomics technique was used. Results revealed that germination percentage, root/ shoot length, lateral roots and fresh biomass were increased in chickpea treated with 2000 ppm plant-derived smoke for 6 days. The number of nodules and rhizobium population were increased. Furthermore, the content of nitrogen and proteins were accumulated. On treatment with 2000 ppm plant-derived smoke for 6 days, the abundance of 90 proteins was significantly changed in chickpea root. Regarding gene ontology (GO) term assignments of 90 proteins related with different biological processes, the proteins related with metabolic process were increased. For cellular process, proteins related with cytoplasm were significantly increased. For molecular function, catalytic activity related proteins were more highly represented than other categories. Proteins related to signaling and transports were increased; however, proteins related to protein metabolism, cell, and cell wall were decreased. The sucrose synthase for starch degradation was increased and total soluble sugar was induced. The proteins for nitrate pathway were increased and nitrate content was improved. On the other hand, although secondary metabolism related proteins were decreased, flavonoid contents were increased. Based on proteomics and immuno-blot analyses, proteins related to redox homeostasis were decreased and increased in root and shoot, respectively. Furthermore, vii fructose-bisphosphate aldolase (FBPA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were increased; while, phosphotransferase and phosphoglycero mutase were decreased in glycolysis. In addition, phosphoglyceraldehyde-3-phosphate dehydrogenase and glutamine synthetase related genes were up-regulated; while, chalcone synthase related genes were down-regulated. These results suggest that plant-derived smoke improves early stage of growth in chickpea with the balance of many cascades such as glycolysis, redox homeostasis and secondary metabolism. This study indicates that the application of plant-derived smoke might be a useful and inexpensive technique for enhancing seedling growth, number of nodules, nutritional values of crops and can be commercialized as bio fertilizer in future.