Plant growth promoting rhizobacterium Stenotrophomonas maltophilia BJ01 augments endurance against N2 starvation by modulating physiology and biochemical activities of Arachis hypogea

English version

Autoři: Ankita Alexander ^aff001; Vijay Kumar Singh ^aff001; Avinash Mishra ^aff001; Bhavanath Jha ^aff001
Působiště autorů: Biotechnology and Phycology Division, CSIR- Central Salt and Marine Chemicals Research Institute, G. B. Marg, Bhavnagar, Gujarat, India ^aff001; Academy of Scientific and Innovative Research (AcSIR), CSIR, Ghaziabad, India ^aff002
Vyšlo v časopise: PLoS ONE 14(9)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0222405

Souhrn

Arachis hypogea (Peanut) is one of the most important crops, and it is harvested and used for food and oil production. Being a legume crop, the fixation of atmospheric nitrogen is achieved through symbiotic association. Nitrogen deficiency is one of the major constrains for loss of crop productivity. The bacterium Stenotrophomonas maltophilia is known for interactions with plants. In this study, characteristics that promote plant growth were explored for their ability to enhance the growth of peanut plants under N₂ deficit condition. In the presence of S. maltophilia, it was observed that fatty acid composition of peanut plants was influenced and increased contents of omega-7 monounsaturated fatty acid and omega-6 fatty acid (γ-Linolenic acid) were detected. Plant growth was increased in plants co-cultivated with PGPR (Plant Growth Promoting Rhizobacteria) under normal and stress (nitrogen deficient) condition. Electrolyte leakage, lipid peroxidation, and H₂O₂ content reduced in plants, co-cultivated with PGPR under normal (grown in a media supplemented with N₂ source; C+) or stress (nitrogen deficient N+) conditions compared to the corresponding control plants (i.e. not co-cultivated with PGPR; C–or N–). The growth hormone auxin, osmoprotectants (proline, total soluble sugars and total amino acids), total phenolic-compounds and total flavonoid content were enhanced in plants co-cultivated with PGPR. Additionally, antioxidant and free radical scavenging (DPPH, hydroxyl and H₂O₂) activities were increased in plants that were treated with PGPR under both normal and N₂ deficit condition. Overall, these results indicate that plants co-cultivated with PGPR, S. maltophilia, increase plant growth, antioxidant levels, scavenging, and stress tolerance under N₂ deficit condition. The beneficial use of bacterium S. maltophilia could be explored further as an efficient PGPR for growing agricultural crops under N₂ deficit conditions. However, a detail agronomic study would be prerequisite to confirm its commercial role.

Klíčová slova:

Biology and life sciences – Organisms – Eukaryota – Plants – Legumes – Peanut – Plant science – Plant physiology – Plant defenses – Plant resistance to abiotic stress – Plant pathology – Plant ecology – Plant-environment interactions – Plant growth and development – Plant anatomy – Leaves – Ecology – Microbiology – Medical microbiology – Microbial pathogens – Bacterial pathogens – Stenotrophomonas maltophilia – Biochemistry – Lipids – Fatty acids – Antioxidants – Developmental biology – Ecology and environmental sciences – Medicine and health sciences – Pathology and laboratory medicine – Pathogens

Zdroje

1. Singh RP, Jha P, Jha PN. Bio-inoculation of plant growth-promoting rhizobacterium Enterobacter cloacae ZNP-3 increased resistance against salt and temperature stresses in wheat plant (Triticum aestivum L.). J Plant Growth Regul. 2017; 36(3):783–98.

2. Patel BB, Patel BB, Dave RS. Studies on infiltration of saline-alkali soils of several parts of Mehsana and Patan districts of North Gujarat. J Appl Technol Environ Sanit. 2011; 1(1):87–92.

3. Munns R, James RA. Screening methods for salinity tolerance: a case study with tetraploid wheat. Plant Soil. 2003; 253(1):201–18.

4. Tester M, Davenport R. Na+ tolerance and Na+ transport in higher plants. Ann Bot. 2003; 91(5):503–27. doi: 10.1093/aob/mcg058 12646496

5. Blaylock AD. Soil salinity, salt tolerance, and growth potential of horticultural and landscape plants. University of Wyoming, Cooperative Extension Service, Department of Plant, Soil, and Insect Sciences, College of Agriculture; 1994.

6. Mishra A, Patel MK, Jha B. Non-targeted metabolomics and scavenging activity of reactive oxygen species reveal the potential of Salicornia brachiata as a functional food. J Funct Food. 2015; 13:21–31.

7. Patel MK, Mishra A, Jha B. 2016. Untargeted metabolomics of halophytes in Marine Omics: Principles and Applications, ed S. Kim (Boca Raton, FL: CRC Press), 309–325. doi: 10.1201/9781315372303-18

8. Mishra A, Joshi M, Jha B. Oligosaccharide mass profiling of nutritionally important Salicornia brachiata, an extreme halophyte. Carbohyd Polym. 2013; 92(2):1942–5.

9. Jha B, Singh NP, Mishra A. Proteome profiling of seed storage proteins reveals the nutritional potential of Salicornia brachiata Roxb., an extreme halophyte. J Agr Food Chem. 2012; 60(17):4320–6.

10. Chaturvedi AK, Mishra A, Tiwari V, Jha B. Cloning and transcript analysis of type 2 metallothionein gene (SbMT-2) from extreme halophyte Salicornia brachiata and its heterologous expression in E. coli. Gene. 2012; 499(2):280–7. doi: 10.1016/j.gene.2012.03.001 22441126

11. Chaturvedi AK, Patel MK, Mishra A, Tiwari V, Jha B. The SbMT-2 gene from a halophyte confers abiotic stress tolerance and modulates ROS scavenging in transgenic tobacco. PloS one. 2014; 9 (10):e111379. doi: 10.1371/journal.pone.0111379 25340650

12. Singh N, Mishra A, Jha B. Over-expression of the peroxisomal ascorbate peroxidase (SbpAPX) gene cloned from halophyte Salicornia brachiata confers salt and drought stress tolerance in transgenic tobacco. Mar Biotechnol. 2014; 16(3):321–32. doi: 10.1007/s10126-013-9548-6 24197564

13. Singh VK, Mishra A, Haque I, Jha B. A novel transcription factor-like gene SbSDR1 acts as a molecular switch and confers salt and osmotic endurance to transgenic tobacco. Sci Rep. 2016; 6:31686. doi: 10.1038/srep31686 27550641

14. Patel MK, Joshi M, Mishra A, Jha B. Ectopic expression of SbNHX1 gene in transgenic castor (Ricinus communis L.) enhances salt stress by modulating physiological process. Plant Cell Tiss Org. 2015; 122(2):477–90.

15. Pandey S, Patel MK, Mishra A, Jha B. In planta transformed cumin (Cuminum cyminum L.) plants, overexpressing the SbNHX1 gene showed enhanced salt endurance. PloS one. 2016; 11(7):e0159349. doi: 10.1371/journal.pone.0159349 27411057

16. Udawat P, Mishra A, Jha B. Heterologous expression of an uncharacterized universal stress protein gene (SbUSP) from the extreme halophyte, Salicornia brachiata, which confers salt and osmotic tolerance to E. coli. Gene. 2014; 536(1):163–70. doi: 10.1016/j.gene.2013.11.020 24291028

17. Udawat P, Jha RK, Sinha D, Mishra A, Jha B. Overexpression of a cytosolic abiotic stress responsive universal stress protein (SbUSP) mitigates salt and osmotic stress in transgenic tobacco plants. Front Plant Sci. 2016; 7:518. doi: 10.3389/fpls.2016.00518 27148338

18. Udawat P, Jha RK, Mishra A, Jha B. Overexpression of a plasma membrane-localized SbSRP-like protein enhances salinity and osmotic stress tolerance in transgenic tobacco. Front Plant Sci. 2017; 8:582. doi: 10.3389/fpls.2017.00582 28473839

19. Mishra A, Tanna B. Halophytes: potential resources for salt stress tolerance genes and promoters. Front Plant Sci. 2017; 8:829. doi: 10.3389/fpls.2017.00829 28572812

20. Jha B, Mishra A, Jha A, Joshi M. Developing transgenic Jatropha using the SbNHX1 gene from an extreme halophyte for cultivation in saline wasteland. PLoS One. 2013; 8(8):e71136. doi: 10.1371/journal.pone.0071136 23940703

21. Jha B, Sharma A, Mishra A. Expression of SbGSTU (tau class glutathione S-transferase) gene isolated from Salicornia brachiata in tobacco for salt tolerance. Mol Biol Rep. 2011; 38(7):4823–32. doi: 10.1007/s11033-010-0625-x 21136169

22. Jha RK, Patel J, Mishra A, Jha B. 2019. Introgression of halophytic salt stress-responsive genes for developing stress tolerance in crop plants. In: Hasanuzzaman M, Shabala S, and Fujita M (Eds.) Halophytes and climate change: adaptive mechanisms and potential uses, CABI, UK, pp. 288–299. doi: 10.1079/9781786394330.0275

23. Tiwari V, Chaturvedi AK, Mishra A, Jha B. The transcriptional regulatory mechanism of the peroxisomal ascorbate peroxidase (pAPX) gene cloned from an extreme halophyte, Salicornia brachiata. Plant Cell Physiol. 2013; 55(1):201–17. doi: 10.1093/pcp/pct172 24285755

24. Tiwari V, Patel MK, Chaturvedi AK, Mishra A, Jha B. Functional characterization of the tau class glutathione-S-transferases gene (SbGSTU) promoter of Salicornia brachiata under salinity and osmotic stress. PLoS One. 2016; 11(2):e0148494. doi: 10.1371/journal.pone.0148494 26885663

25. Tiwari V, Patel MK, Chaturvedi AK, Mishra A, Jha B. Cloning and functional characterization of the Na+/H+ antiporter (NHX1) gene promoter from an extreme halophyte Salicornia brachiata. Gene. 2019; 683:233–42. doi: 10.1016/j.gene.2018.10.039 30340051

26. Patel MK, Pandey S, Brahmbhatt HR, Mishra A, Jha B. Lipid content and fatty acid profile of selected halophytic plants reveal a promising source of renewable energy. Biomass and Bioenergy. 2019; 124, 25–32.

27. Alexander A, Mishra A, Jha B. 2019. Halotolerant Rhizobacteria: A Promising Probiotic for Saline Soil-Based Agriculture. In: Kumar M., Etesami H., Kumar V. (eds) Saline Soil-based Agriculture by Halotolerant Microorganisms. Springer, Singapore, pp. 53–73. https://doi.org/10.1007/978-981-13-8335-9_3

28. Singh VK, Kavita K, Prabhakaran R, Jha B. Cis-9-octadecenoic acid from the rhizospheric bacterium Stenotrophomonas maltophilia BJ01 shows quorum quenching and anti-biofilm activities. Biofouling. 2013; 29(7):855–67. doi: 10.1080/08927014.2013.807914 23844805

29. Ryan RP, Monchy S, Cardinale M, Taghavi S, Crossman L, Avison MB, Berg G, Van Der Lelie D, Dow JM. The versatility and adaptation of bacteria from the genus Stenotrophomonas. Nat Rev Microbiol. 2009; 7(7):514. doi: 10.1038/nrmicro2163 19528958

30. Singh VK, Mishra A, Jha B. Anti-quorum sensing and anti-biofilm activity of Delftia tsuruhatensis extract by attenuating the quorum sensing-controlled virulence factor production in Pseudomonas aeruginosa. Front Cell Infect Microbiol. 2017; 7:337. doi: 10.3389/fcimb.2017.00337 28798903

31. Singh VK, Mishra A, Jha B. 3-Benzyl-hexahydro-pyrrolo [1, 2-a] pyrazine-1, 4-dione extracted from Exiguobacterium indicum showed anti-biofilm activity against Pseudomonas aeruginosa by attenuating quorum sensing. Frontiers in Microbiology, 2019; 10:1269. doi: 10.3389/fmicb.2019.01269 31231348

32. Singh RP, Jha P N. The PGPR Stenotrophomonas maltophilia SBP-9 augments resistance against biotic and abiotic stress in wheat plants. Frontiers in Microbiology, 2017, 8, 1945. doi: 10.3389/fmicb.2017.01945 29062306

33. Pandey MK, Monyo E, Ozias-Akins P, Liang X, Guimarães P, Nigam SN, Upadhyaya HD, Janila P, Zhang X, Guo B, Cook DR. Advances in Arachis genomics for peanut improvement. Biotechnol Adv. 2012; 30(3):639–51. doi: 10.1016/j.biotechadv.2011.11.001 22094114

34. Singh N, Mishra A, Jha B. Ectopic over-expression of peroxisomal ascorbate peroxidase (SbpAPX) gene confers salt stress tolerance in transgenic peanut (Arachis hypogaea). Gene. 2014; 547(1):119–25. doi: 10.1016/j.gene.2014.06.037 24954532

35. Tiwari V, Chaturvedi AK, Mishra A, Jha B. An efficient method of Agrobacterium-mediated genetic transformation and regeneration in local Indian cultivar of groundnut (Arachis hypogaea) using grafting. Appl Biochem Biotech. 2015; 175(1):436–53.

36. Tiwari V, Chaturvedi AK, Mishra A, Jha B. Introgression of the SbASR-1 gene cloned from a halophyte Salicornia brachiata enhances salinity and drought endurance in transgenic groundnut (Arachis hypogaea) and acts as a transcription factor. PLoS One. 2015; 10(7):e0131567. doi: 10.1371/journal.pone.0131567 26158616

37. Jha B, Mishra A, Chaturvedi AK. Engineering Stress Tolerance in Peanut (Arachis hypogaea L.). In: Watson R, Preedy VR(Eds.) Genetically Modified Organisms (GMO) Foods: Production, Regulation and Public Health 2016 (pp. 305–311). Elsevier, Philadelphia, USA.

38. Yousuf B, Keshri J, Mishra A, Jha B. Application of targeted metagenomics to explore abundance and diversity of CO2-fixing bacterial community using cbbL gene from the rhizosphere of Arachis hypogaea. Gene. 2012; 506(1):18–24. doi: 10.1016/j.gene.2012.06.083 22766402

39. Keshri J, Mishra A, Jha B. Microbial population index and community structure in saline–alkaline soil using gene targeted metagenomics. Microbiological Research, 2013; 168(3):165–173. doi: 10.1016/j.micres.2012.09.005 23083746

40. Arnon DI. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant physiol. 1949; 24(1):1. 16654194

41. Chamovitz D, Sandmann G, Hirschberg J. Molecular and biochemical characterization of herbicide-resistant mutants of cyanobacteria reveals that phytoene desaturation is a rate-limiting step in carotenoid biosynthesis. J Biol Chem. 1993; 268(23):17348–53. 8349618

42. Lutts S, Kinet JM, Bouharmont J. NaCl-induced senescence in leaves of rice (Oryza sativa L.) cultivars differing in salinity resistance. Ann Bot. 1996; 78(3):389–98.

43. Hayat S, Yadav S, Ali B, Ahmad A. Interactive effect of nitric oxide and brassinosteroids on photosynthesis and the antioxidant system of Lycopersicon esculentum. Russ J Plant Physl. 2010; 57(2):212–21.

44. Hodges DM, DeLong JM, Forney CF, Prange RK. Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta. 1999; 207(4):604–11.

45. Mukherjee SP, Choudhuri MA. Implications of water stress‐induced changes in the levels of endogenous ascorbic acid and hydrogen peroxide in Vigna seedlings. Physiol Plantarum. 1983; 58(2):166–70.

46. Andreae WA, Van Ysselstein MW. Studies on 3-indoleacetic acid metabolism. V. Effect of calcium ions on 3-indoleacetic acid uptake and metabolism by pea roots. Plant physiol. 1960; 35(2):220. 16655332

47. Bates LS, Waldren RP, Teare ID. Rapid determination of free proline for water-stress studies. Plant soil. 1973; 39(1):205–7.

48. Patel MK, Mishra A, Jha B. Non-targeted metabolite profiling and scavenging activity unveil the nutraceutical potential of psyllium (Plantago ovata Forsk). Frontiers in Plant Science. 2016; 7:431. doi: 10.3389/fpls.2016.00431 27092153

49. Pandey S, Patel MK, Mishra A, Jha B. Physio-biochemical composition and untargeted metabolomics of cumin (Cuminum cyminum L.) make it promising functional food and help in mitigating salinity stress. PLoS One, 2015; 10(12):e0144469. doi: 10.1371/journal.pone.0144469 26641494

50. Hazra B, Biswas S, Mandal N. Antioxidant and free radical scavenging activity of Spondias pinnata. BMC Complem Altern M. 2008; 8(1):63.

51. Tanna B, Choudhary B, Mishra A. Metabolite profiling, antioxidant, scavenging and anti-proliferative activities of selected tropical green seaweeds reveal the nutraceutical potential of Caulerpa spp. Algal Res. 2018; 36:96–105.

52. Zhishen J, Mengcheng T, Jianming W. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem. 1999; 64(4):555–9.

53. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free radical Bio Med. 1999; 26(9–10):1231–7.

54. Saeed N, Khan MR, Shabbir M. Antioxidant activity, total phenolic and total flavonoid contents of whole plant extracts Torilis leptophylla L. BMC Complem Altern M. 2012; 12(1):221.

55. Yousuf B, Kumar R, Mishra A, Jha B. Differential distribution and abundance of diazotrophic bacterial communities across different soil niches using a gene-targeted clone library approach. FEMS Microbiol Lett. 2014; 360(2):117–25. doi: 10.1111/1574-6968.12593 25196726

56. Keshri J, Yousuf B, Mishra A, Jha B. The abundance of functional genes, cbbL, nifH, amoA and apsA, and bacterial community structure of intertidal soil from Arabian Sea. Microbiol Res. 2015; 175:57–66. doi: 10.1016/j.micres.2015.02.007 25862282

57. Kanehara S, Ohtani T, Uede K, Furukawa F. Clinical effects of undershirts coated with borage oil on children with atopic dermatitis: A double‐blind, placebo‐controlled clinical trial. The J Dermatol. 2007; 34(12):811–5. 18078406

58. Dubois V, Breton S, Linder M, Fanni J, Parmentier M. Fatty acid profiles of 80 vegetable oils with regard to their nutritional potential. Eur J Lipid Sci Tech. 2007; 109(7):710–32.

59. Zheng CJ, Yoo JS, Lee TG, Cho HY, Kim YH, Kim WG. Fatty acid synthesis is a target for antibacterial activity of unsaturated fatty acids. FEBS Lett. 2005; 579(23):5157–62. 16146629

60. Silva LR, Pereira MJ, Azevedo J, Mulas R, Velazquez E, González-Andrés F, Valentão P, Andrade PB. Inoculation with Bradyrhizobium japonicum enhances the organic and fatty acids content of soybean (Glycine max (L.) Merrill) seeds. Food Chem. 2013; 141(4):3636–48. doi: 10.1016/j.foodchem.2013.06.045 23993531

61. Brechenmacher L, Lei Z, Libault M, Findley S, Sugawara M, Sadowsky MJ, Sumner LW, Stacey G. Soybean metabolites regulated in root hairs in response to the symbiotic bacterium Bradyrhizobium japonicum. Plant Physiol. 2010; 153(4):1808–22. doi: 10.1104/pp.110.157800 20534735

62. Cagide C, Riviezzi B, Minteguiaga M, Morel MA, Castro-Sowinski S. Identification of Plant Compounds Involved in the Microbe-Plant Communication During the Coinoculation of Soybean with Bradyrhizobium elkanii and Delftia sp. strain JD2. Mol Plant Microbe In. 2018; 31(11):1192–9.

63. Morel MA, Cagide C, Minteguiaga MA, Dardanelli MS, Castro-Sowinski S. The pattern of secreted molecules during the co-inoculation of alfalfa plants with Sinorhizobium meliloti and Delftia sp. strain JD2: an interaction that improves plant yield. Mol Plant Microbe In. 2015; 28(2):134–42.

64. Morel MA, Braña V, Castro-Sowinski S. Legume crops, importance and use of bacterial inoculation to increase production. InCrop plant 2012. IntechOpen.

65. Ripullone F, Grassi G, Lauteri M, Borghetti M. Photosynthesis–nitrogen relationships: interpretation of different patterns between Pseudotsuga menziesii and Populus euroamericana in a mini-stand experiment. Tree Physiol. 2003; 23(2):137–44. 12533308

66. Nisar N, Li L, Lu S, Khin NC, Pogson BJ. Carotenoid metabolism in plants. Mol Plant. 2015; 8(1):68–82. doi: 10.1016/j.molp.2014.12.007 25578273

67. Bashan Y, Bustillos JJ, Leyva LA, Hernandez JP, Bacilio M. Increase in auxiliary photoprotective photosynthetic pigments in wheat seedlings induced by Azospirillum brasilense. Biol Fert Soils. 2006; 42(4):279–85.

68. Cohen AC, Bottini R, Pontin M, Berli FJ, Moreno D, Boccanlandro H, Travaglia CN, Piccoli PN. Azospirillum brasilense ameliorates the response of Arabidopsis thaliana to drought mainly via enhancement of ABA levels. Physiol Plantarum. 2015; 153(1):79–90.

69. Claussen W. Proline as a measure of stress in tomato plants. Plant Sci. 2005; 168(1):241–8.

70. Koca H, Ozdemir F, Turkan I. Effect of salt stress on lipid peroxidation and superoxide dismutase and peroxidase activities of Lycopersicon esculentum and L. pennellii. Biol Plantarum. 2006; 50(4):745–8.

71. Yazici I, Türkan I, Sekmen AH, Demiral T. Salinity tolerance of purslane (Portulaca oleracea L.) is achieved by enhanced antioxidative system, lower level of lipid peroxidation and proline accumulation. Environ Exp Bot. 2007; 61(1):49–57.

72. Tanna B, Mishra A. Metabolites unravel nutraceutical potential of edible seaweeds: an emerging source of functional food. Comprehensive Reviews in Food Science and Food Safety, 2018; 17(6):1613–1624.

73. Seneviratne G, Jayasinghearachchi HS. Phenolic acids: Possible agents of modifying N 2-fixing symbiosis through rhizobial alteration?. Plant soil. 2003; 252(2):385–95.

74. Makoi JH, Ndakidemi PA. Biological, ecological and agronomic significance of plant phenolic compounds in rhizosphere of the symbiotic legumes. Afr J Biotechnol. 2007; 6(12).

75. Juge C, Prévost D, Bertrand A, Bipfubusa M, Chalifour FP. Growth and biochemical responses of soybean to double and triple microbial associations with Bradyrhizobium, Azospirillum and arbuscular mycorrhizae. Appl Soil Ecol. 2012; 61:147–57.

76. Antunes PM, Rajcan I, Goss MJ. Specific flavonoids as interconnecting signals in the tripartite symbiosis formed by arbuscular mycorrhizal fungi, Bradyrhizobium japonicum (Kirchner) Jordan and soybean (Glycine max (L.) Merr.). Soil Biol Biochem. 2006; 38(3):533–43.

77. Chang C, Damiani I, Puppo A, Frendo P. Redox changes during the legume–Rhizobium symbiosis. Mol Plant. 2009; 2(3):370–7. doi: 10.1093/mp/ssn090 19825622

78. Prakash D, Singh BN, Upadhyay G. Antioxidant and free radical scavenging activities of phenols from onion (Allium cepa). Food chem. 2007; 102(4):1389–93.

79. Kim JA, Jung WS, Chun SC, Yu CY, Ma KH, Gwag JG, Chung IM. A correlation between the level of phenolic compounds and the antioxidant capacity in cooked-with-rice and vegetable soybean (Glycine max L.) varieties. Eur Food Res Technol. 2006; 224(2):259–70.

80. Sakthivelu G, Akitha Devi MK, Giridhar P, Rajasekaran T, Ravishankar GA, Nikolova MT, Angelov GB, Todorova RM, Kosturkova GP. Isoflavone composition, phenol content, and antioxidant activity of soybean seeds from India and Bulgaria. J Agr Food Chem. 2008; 56(6):2090–5.

Plant growth promoting rhizobacterium Stenotrophomonas maltophilia BJ01 augments endurance against N2 starvation by modulating physiology and biochemical activities of Arachis hypogea

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