Elsevier

Phytochemistry

Volume 179, November 2020, 112506
Phytochemistry

Biochemical and molecular insights of PGPR application for the augmentation of carotenoids, tocopherols, and folate in the foliage of Moringa oleifera

https://doi.org/10.1016/j.phytochem.2020.112506Get rights and content

Highlights

  • B. pumilus T4 & P. fluorescens UOM14 significantly enhanced α-tocopherol.

  • B. subtilis GB03 & P. fluorescens UOM14 significantly enhanced β-carotene.

  • B. subtilis IN937 B, B. pumilus SE34 & T4 showed significant increase in folate.

  • Combination containing all organisms from B. pumilus performed significantly.

  • PGPR's perform significantly in combination compared to individual treatment.

Abstract

Plant Growth Promoting Rhizobacteria (PGPR) were utilized to contemplate their impact on the foliage of Moringa oleifera and examined for changes in tocopherols, chlorophyll, carotenoids, and folate in the sixth week. Among the eight treatments, Bacillus subtilis GB03, B. pumilus SE34, B. pumilus T4, and Pseudomonas fluorescens UOM14 improved α-tocopherol (10–14 fold) and β-carotene (1–1.40 fold) altogether significantly (P ≤ 0.05). The most significant improvement in folate content was apparent for B. subtilis IN937B (5.47 fold) trailed by B. pumilus SE34 (5.05 fold) and B. pumilus T4 (5.12 fold) treatments. P. fluorescens UOM14 indicated remarkable improvement in Chl a (0.39 fold) and Chl b (0.44 fold) content. Organisms showing a significant increase for the analyzed molecules in individual treatment were blended in different combinations and were used for the next set of treatments. Of all the three combinations, Combination 2 (COM2-B. pumilus SE34 + B. pumilus T4 + B. pumilus INR7) showed the maximum increase in α-tocopherol (8.46 fold) and γ-tocopherol (8.45 fold), followed by Combination 3 (COM3-B. pumilus SE34 + B. pumilus T4 + P. fluorescens UOM14) (5.93 and 3.65 fold). On the whole COM2 containing different strains of B. pumilus was found to enhance the targeted metabolites in foliage significantly. Real-time PCR studies were conducted for the biochemical pathway genes of the targeted molecules, including, γ-tocopherol methyltransferase (γ-TMT), phytoene synthase (PSY), phytoene desaturase (PDS), lycopene β cyclase (LBC) and dihydrofolate reductase thymidylate synthase (DHFR-TS). All the selected genes exhibited an up-regulation compared to control, similar to the biochemical output. Our investigation provides the strong evidence that PGPR can be viably utilized in combination to enhance the quality of the food crops.

Introduction

Plant growth-promoting rhizobacteria (PGPR) are among the best possible alternatives to decrease the rampant use of chemical fertilizers and to increase agricultural productivity (Srivastava and Singh, 2019). PGPR are known to improve plant growth by adopting different biochemical, molecular and physiological pathways. PGPR stimulate plant growth by fixing nitrogen, solubilizing phosphorous, producing vitamins and phytohormones such as auxins, gibberellins, cytokinins. PGPR are well known biotic elicitors acting through different signal perception like jasmonic acid (JA), ethylene (ET) etc. activating the wide array of defence-related compounds and enhances the production of secondary metabolites (Govindasamy et al., 2010; Pradeep and Giridhar, 2017). Different bacteria's like Azoarcus, Azospirillum, Azotobacter, Arthrobacter, Bacillus, Clostridium, Enterobacter, Gluconacetobacter, Pseudomonas, and Serratia were found to exhibit PGPR activity. Of all the above mentioned organisms, different strains of Pseudomonas and Bacillus have been widely exploited for their growth promoting activities on plants. Bacillus is common inhabitant of plants and soil as they survive under adverse agricultural and environmental conditions (Borriss, 2011; Kumar et al., 2011). Pseudomonas fluorescens also show easy adaptation and strong survival in soil. They are metabolically and functionally more diverse which helps them to maintain soil health. Bacillus and P. fluorescens were reported to be promising biocontrol, biofertiliser, growth promoter and minerals enhancer because of its ability to act as bio-elicitor (Borriss, 2011; Kumar et al., 2011; Priyanka et al., 2017; Rosier et al., 2018).

Enhanced synthesis of complicated vitamins after PGPR application on the plant has raised the question about its involvement in important steps during plant PGPR interaction. During the interaction, vitamins produced by plant or bacteria and act as co-factors for different biochemical pathways, promote plant growth, induce systemic resistance, engage in the production of essential compounds for plants and bacteria. Augmentation of most of the B-complex vitamins such as thiamine, riboflavin and niacin during plant-microbe interaction has been studied (Burgess et al., 2009; Palacios et al., 2014). Amongst the B-complex vitamins, folate is not widely studied but is suspected to be produced mainly by the plant during plant-microbe interaction (Burgess et al., 2009; Palacios et al., 2014). At present, little is known about the antioxidant activity of folate and pterin in plants but some studies show enhancement of folate using plant stress hormones such as methyl jasmonate, salicylic acid, and abscisic acid which hints toward its linkage to stress pathways (Puthusseri et al., 2012). PGPR is also known to produce stress hormones during localization; hence its application can lead to increase in the levels of anti-oxidative molecules like tocopherols, carotenoids, chlorophyll and folate which are amongst the major antioxidants in plants (Barickman et al., 2014). Vitamin A, Vitamin E and folate produced in plants are of great human importance. Their deficiencies are prevalent in the impoverished population and developing countries. Vitamins act as an antioxidant agent, pro-oxidant, cell signaling and gene regulatory molecule (Kurutas, 2016). They prevent and treat atherosclerosis, cardiovascular complications, cancer, Alzheimer's, neural tube defects and many other diseases in humans (Tam et al., 2013; Kurutas, 2016). They are involved in the synthesis of nucleic acids, methionine, pantothenate, glycine, and serine (Miret and Munné-Bosch, 2014).

PGPR acts as a biotic elicitor leading to an increase in reactive oxygen species (ROS). Along with the increase in ROS, many reports also state the release of stress hormones like salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and abscisic acid (ABA). An increase in stress hormones will trigger the overexpression of many pathway leading to a change in the metabolite level to counteract stress and to decrease ROS (Barickman et al., 2014). Direct application of SA, JA, ABA was reported to elicit the formation of tocopherols, carotenoids, folates in many in vitro studies (Puthusseri et al., 2012; Saini et al., 2014b). Many PGPR studies have concentrated on enhancing the quantity of crops, but to date, no report states its importance for enhancing the quality of the crop.

Moringa oleifera Lam, (Moringaceae) is known for its astounding content of many nutrients and can be used as a nutrition security provider in many underdeveloped or developing countries (Saini et al., 2013). Recent studies on this plant have demonstrated wide genetic diversity among commonly grown cultivars (Saini et al., 2014a). In addition, its foliage richness in carotenoids and fatty acid augmentation using abiotic elicitor to enhance carotenoids and tocopherols and bioavailability of iron and folate from M. oleifera in rat models was reported (Saini et al., 2014b, 2016). To make it a more capable nutritional security provider, M. oleifera was chosen for the study. Accordingly, PGPR were used for the study to find out their influence on total carotenoids, β-carotene, tocopherols, and folate in M. oleifera and for a profile enrichment of same for value addition.

Section snippets

Results and discussion

M. oleifera plants were treated with individual PGPR (Fig. 1). The sampling time was standardized post treatment at the 6th week based on several preliminary studies. After sampling, tocopherols, carotenoids, chlorophylls, and folates analysis was executed. PGPR showing significant enhancement in the metabolites were selected for combination studies. Before combining the organisms, they were tested for compatibility. The main aim of the combination study was to see the synergistic effects of

Conclusions

In the current investigation, external PGPR were utilized notwithstanding the presence of native endophytes. PGPR which were utilized in the investigation were seen to advance the development and production of notable nutrients of human importance. This investigation demonstrates that the inoculation of PGPR emphatically improves tocopherols, carotenoids, and folate in the foliage of the plant. PGPR performing significantly in individual treatments were studied into different combinations.

General experimental procedure

All the chemicals and kits like standards of carotenoids, chlorophyll, tocopherols, Folate binding protein, α-amylase from Aspergillus oryzae (30 units/mg) and protease from Streptomyces griseus (Type XIV, 3.5 units/mg), Spectrum Plant total RNA kit were purchased from Merck, Bengaluru, India whereas 5,6,7,8 Tetrahydro folic acid standard was purchased from Schircks Laboratories (Jona, Switzerland). Affigel matrix was purchased from BIO-RAD (Gurgaon, India). Maxima SYBR green/ROX master mix,

Funding

This work was supported by grants from the Council of Scientific and Industrial Research, New Delhi (CSIR-CFTRI-MLP0152) and furthermore giving fellowship to PPS, and KK.

Declaration of competing interest

The authors have no conflicts of interest to declare.

Acknowledgments

The authors are grateful to Dr. Niranjan Raj of the University of Mysore for providing bacterial PGPR.

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