Date Published: March 4, 2019
Publisher: Public Library of Science
Author(s): Naeem Khan, Asghari Bano, MD Ali Babar, Prasanta K. Subudhi.
Plant growth regulators (PGRs) and plant growth promoting rhizobacteria (PGPRs) play an important role in mitigating abiotic stresses. However, little is known about the parallel changes in physiological processes coupled with metabolic changes induced by PGRs and PGPRs that help to cope with drought stress in chickpeas. The present investigation was carried out to study the integrative effects of PGRs and PGPRs on the physiological and metabolic changes, and their association with drought tolerance in two chickpea genotypes. Inoculated seeds of two chickpea genotypes, Punjab Noor-2009 (drought sensitive) and 93127 (drought tolerance), were planted in greenhouse condition at the University of Florida. Prior to sowing, seeds of two chickpea varieties were soaked for 3 h in 24 h old cultures of PGPRs (Bacillus subtilis, Bacillus thuringiensis, and Bacillus megaterium), whereas, some of the seeds were soaked in distilled water for the same period of time and were treated as control. Plant growth regulators, salicylic acid (SA) and putrescine (Put), were applied on 25 days old seedlings just prior to the induction of drought stress. Drought stress was imposed by withholding the supply of water on 25-day-old seedlings (at the three-leaf stage) and continued for the next 25 days until the soil water content reached 14%. Ultrahigh-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) analysis concomitant with physiological parameters were carried out in chickpea leaves at two-time points i.e. 14 and 25 d after imposition of drought stress. The results showed that both genotypes, treated with PGRs and PGPRs (consortium), performed significantly better under drought condition through enhanced leaf relative water content (RWC), greater biomass of shoot and root, higher Fv/FM ratio and higher accumulation of protein, sugar and phenolic compounds. The sensitive genotype was more responsive than tolerant one. The results revealed that the accumulation of succinate, leucine, disaccharide, saccharic acid and glyceric acid was consistently higher in both genotypes at both time points due to PGRs and PGPRs treatment. Significant accumulation of malonate, 5-oxo-L-proline, and trans-cinnamate occurred at both time points only in the tolerant genotype following the consortium treatment. Aminoacyl-tRNA, primary and secondary metabolite biosynthesis, amino acid metabolism or synthesis pathways, and energy cycle were significantly altered due to PGRs and PGPRs treatment. It is inferred that changes in different physiological and metabolic parameters induced by PGRs and PGPRs treatment could confer drought tolerance in chickpeas.
Chickpea (Cicer arietinum L.) is a legume belongs in the family) Fabaceae, subfamily Faboideae. It is one of the most widely consumed pulse legumes and ranking third after peas and soybean, and also covers a total of 15% of the world’s pulse productions . It is an important source of protein, carbohydrate, B-group vitamins, and different minerals . It is considered an important source of cheap protein with high energy and nutritional values [3, 4]. Drought stress is the most prevalent environmental factor that limits growth, survival, and productivity of chickpeas . The yield of chickpeas can be reduced from 15 to 60% due to the drought stress. Moreover, the global climate change including high temperature stress and unpredictable rainfall pattern coupled with the increasing world population is creating immense pressure on our capacity for sustainable food production including chickpea production. Drought affects seed germination and seedling establishment in the field, however, genotypes vary in their capacity to tolerate drought stress. Drought also causes a substantial reduction in crop productivity through negatively impacting plant growth, physiology, nutrient and water relations, photosynthesis, and assimilate partitioning [6–8]. To cope with such challenges and develop stress resilient chickpea varieties for future climate change condition, understanding the effects of drought on physiological, morphological and biochemical processes, and their relationship to the adaptation mechanisms is crucial .
The experiment was conducted under greenhouse condition at the Department of Agronomy, University of Florida, Gainesville, Florida in May, 2016. Seeds of two chickpea genotypes, Punjab Noor-2009 (drought sensitive) and 93127 (drought tolerance), were obtained from Ayub Agriculture Research Institute, Faisalabad, Pakistan. Initially, chickpea seeds were washed in distilled water followed by surface sterilization with 95% ethanol for 2–3 min and then soaked in 10% Clorox with concomitant shaking. The seeds were subsequently washed in autoclaved distilled water. Some of the washed seeds were soaked for 3 h, prior to sowing, in 24 h old cultures of Bacillus subtilis, Bacillus thuringiensis, and Bacillus megaterium, whereas, some of the washed seeds were sown without any treatment (drought control). The PGRs, salicylic acid (SA) and putrescine (Put) were sprayed (150 mg/L) on 25 days old seedlings of chickpea inoculated with PGPRs. Seeds were grown in pots (5 seeds/pot) measuring 30 × 40 cm2 and filled with 2,000 g of Metro-Mix 360 soil mixture. The pots were well watered (twice per week) throughout until drought stress was applied, and each pot contained 5 plants. Water was applied until the soil mix was completely wet and the water started to seep out through the holes at the bottom of the pot. A teaspoon of Osmocote (15N–9P–12K) was applied one time after germination. The green house condition was maintained at 26 and 19 ± 1 °C (day and night temperatures) with 70 ± 2% relative humidity and 11 and 13 hours day and night lengths, respectively. Drought stress was imposed on 25-day-old plants (at the 3-leaf stage) by withholding water supply for the next 25 days until the soil water content reached 14%. Leaf tissue samples were collected twice; at 14 days (first-time point) and 25 days (second-time point) after drought stress initiated for metabolomics analysis and different physiological trait estimation.
Drought stress is one of the major constraints for agricultural productivity throughout the world. Approximately, 40% of the agricultural lands are located in the arid and semi-arid regions of the world  where plants suffer frequently from drought stress. Both plant hormones (PGRs) and PGPRs respond to environmental stresses and impart tolerance to plant against the stresses . PGRs and PGPRs consortium plays a significant role in the alleviation of drought stress in plants by maintaining water budget of the plant and by producing metabolites as observed during the present investigation. In the sensitive genotype, the ameliorating effect of PGRs and PGPRs consortium was noteworthy. Chlorophyll content, chlorophyll fluorescence, RWC, and root and shoot biomass accumulation were greater than 2 fold due to PGRs and PGPRs consortium under drought stress. The effect of PGRs and PGPRs treatment was more pronounced in the sensitive genotype compared to the tolerant genotype. Previous studies demonstrated that the drought-induced reduction in chlorophyll content and chlorophyll fluorescence led to a decrease in photosynthesis and overall plant growth [55, 56]. The combined treatment significantly enhanced the chlorophyll fluorescence and chlorophyll content values in the PGRs/PGPRs treated plants in both genotypes. The application of PGRs significantly enhanced the root and shoot growth, and dry matter production in different plants [42, 57]. Similarly, PGPRs are known to enhance the root growth and uptake of minerals and water, thus promote the growth of the whole plant which in turn has a positive impact on plant dry matter content in wheat  and in chickpea . Hassanzadeh et al.  reported that decrease in RWC is related to the decrease in chlorophyll content and leaf fresh weight in sesame genotypes, however, the tolerant genotypes maintained higher RWC under stress condition and thus showed higher affinity for chlorophyll content and leaf fresh weight. Our results demonstrated that PGRs combined with PGPRs helped both genotypes to maintain an efficient photosystem with improved water budget resulting in improved growth and productivity under drought stress condition.
Different amino acids, sugars, sugar alcohol, amines, organic acids, fatty acids and other intermediate compounds were changed significantly due to PGRs and PGPRs treatment. Similar to physiological responses, sensitive genotype also showed altered levels of more metabolites than tolerant genotype. The accumulation of succinate, leucine, disaccharide, saccharic aid and glyceric acid was significantly higher in both genotypes in both time points due to PGRs and PGPRs treatment. As these metabolite levels were constantly higher in both genotypes and at different time points, demonstrating their roles in monitoring biochemical pathways related to drought tolerance. Significant accumulation of malonate, 5-oxo-L-proline, and trans-cinnamate occurred at both time points only in the tolerant genotype due to the consortium treatment. On the contrary, lactic acid, L-carnitine, isocytosine, and phenylpyruvate were accumulated significantly in sensitive genotypes at both times. These results indicate that the higher accumulation of these metabolites could possibly associated only with the tolerance mechanism in sensitive genotype. These data provide information that may, with further investigation, help to understand the biochemical pathway underlying drought stress tolerance in chickpea induced by PGRs and PGPRs treatment.