Acta Physiologiae Plantarum

, 41:152 | Cite as

Phospholipase C activity is increased in wheat seedlings inoculated with the rhizobacteria Azospirillum brasilense Sp245

  • Elda Castro-Mercado
  • Ernesto García-PinedaEmail author
Original Article


The present study aimed to determine phospholipase C (PLC) activation status during changes promoted by Azospirillum brasilense Sp245 in wheat seedlings. Germinated Triticum aestivum seeds by 3 days were inoculated with different A. brasilense concentrations (1 × 106 and 4 × 106 CFU/mL) and PLC enzyme activity assayed at different times. Root and leaf length, as well as total fresh weight were assessed as growth parameters; moreover, changes in root morphology were analyzed. PLC activity was measured by molybdate assay. Neomycin and LaCI3 treatments verified PLC- and/or Ca2+-dependent effects on inoculated wheat, respectively. A. brasilense increased PLC activity 15–30 min after inoculation. Ca2+-channel blocker LaCI3 decreased PLC activity, and activity did not recover with A. brasilense. The A. brasilense mutant FAJ009, impaired in auxin production, showed decreased PLC activity versus wild type. PLC activity was inhibited by neomycin (PLC inhibitor), concomitant with a decrease in total fresh weight. Exogenous addition of diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP3), secondary messengers produced by the activity of PLC, increased root hair length. The putative role of PLC enzyme activity in morphological changes promoted by A. brasilense in wheat seedlings is discussed.


Azospirillum brasilense Wheat Phospholipase C Plant growth promotion Neomycin 



The authors are grateful to Dr. Gladys Alexandre (Tennessee University) for revising and rewriting the manuscript. This study was supported by the Coordinación de la Investigación Científica, Universidad Michoacana de San Nicolás de Hidalgo, México.

Compliance with ethical standards

Conflict of interest

The authors declare that there are no conflicts of interests.


  1. Andreeva Z, Barton D, Armour WJ, Li MY, Liao LF, McKellar HL, Pethybridge KA, Marc J (2010) Inhibition of phospholipase C disrupts cytoskeletal organization and gravitropic growth in Arabidopsis roots. Plant 232:1263–1279. CrossRefGoogle Scholar
  2. Baldani VLD, de Alvarez MA MAB, Baldani JI, Dobereiner JD (1986) Establishment of inoculated Azospirillum spp. in the rhizosphere and in roots of field grown wheat and sorghum. Plant Soil 90:35–46. CrossRefGoogle Scholar
  3. Bashan Y, de-Bashan L (2010) How the plant growth-promoting bacterium Azospirillum promotes plant growth. A critical assessment. Adv Agron 108:77–136. CrossRefGoogle Scholar
  4. Bashan Y, Holguin G, de-Bashan LE (2004) Azospirillum–plant relationships: physiological, molecular, agricultural, and environmental advances (1997–2003). Can J Microbiol 50:521–577. CrossRefGoogle Scholar
  5. Berridge MJ (1993) Inositol trisphosphate and calcium signaling. Nature 361:315–325. CrossRefPubMedGoogle Scholar
  6. Canonne J, Froidure-Nicolas S, Rivas S (2011) Phospholipases in action during plant defense signaling. Plant Signal Behav 6:13–18. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Cassan F, Diaz-Zorita M (2016) Azospirillum sp. in current agriculture: from the laboratory to the field. Soil Biol Biochem 103:117–130. CrossRefGoogle Scholar
  8. Chapman KD (1998) Phospholipase activity during plant growth and development and in response to environmental stress. Trends Plant Sci 3:411–426. CrossRefGoogle Scholar
  9. Chen G, Snyder CL, Greer MS, Weselake RJ (2011) Biology and biochemistry of plant phospholipases. Crit Rev Plant Sci 30:239–258. CrossRefGoogle Scholar
  10. Dowd PE, Coursol S, Skirpan AL, Kao TH, Gilroy S (2006) Petunia phospholipase c1 is involved in pollen tube growth. Plant Cell 18:1438–1453. CrossRefPubMedPubMedCentralGoogle Scholar
  11. Durban MA, Bornscheuer UT (2007) An improved assay for the determination of phospholipase C activity. Eur J Lipid Sci Technol 109:469–473. CrossRefGoogle Scholar
  12. Gabev E, Kasianowicz J, Abbott T, McLaughlin S (1989) Binding of neomycin to phosphatidylinositol 4,5-bisphosphate (PIP 2). Biochim Biophys Acta 979:105–112. CrossRefPubMedGoogle Scholar
  13. Hirayama T, Ohto C, Mizoguchi T, Shinozaki K (1995) A gene encoding a phosphatidylinositol-specific phospholipase C is induced by dehydration and salt stress in Arabidopsis thaliana. Proc Natl Acad Sci USA 92:3903–3907CrossRefGoogle Scholar
  14. Hong Y, Zhao J, Guo L, Kim S-C, Deng X, Wang G, Zhang G, Li M, Wang X (2016) Plant phospholipases D and C and their diverse functions in stress responses. Prog Lipid Res 62:55–74. CrossRefPubMedGoogle Scholar
  15. Kanehara K, Yu C-Y, Cho Y, Cheong W-F, Torta F, Shui G, Wenk MR, Nakamura Y (2015) Arabidopsis AtPLC2 is a primary phosphoinositide-specific phospholipase C in phosphoinositide metabolism and the endoplasmic reticulum stress response. PLoS Genet 11:e1005511. CrossRefPubMedPubMedCentralGoogle Scholar
  16. Khodakovskaya M, Sword C, Wu Q, Perera IY, Boss WF, Brown CS, Sederoff HW (2010) Increasing inositol (1,4,5)-trisphosphate metabolism affects drought tolerance, carbohydrate metabolism and phosphate-sensitive biomass increases in tomato. Plant Biotechnol J 8:170–183. CrossRefPubMedGoogle Scholar
  17. Komis G, Galatis B, Quader H, Galanopoulou D, Apostolakos P (2008) Phospholipase C signaling involvement in macrotubule assembly and activation of the mechanism regulating protoplast volume in plasmolyzed root cells of Triticum turgidum. New Phytol 178:267–282. CrossRefPubMedGoogle Scholar
  18. Mariani ME, Madoery RR, Fidelio GD (2015) Auxins action on Glycine max secretory phospholipase A2 is mediated by the interfacial properties imposed by the phytohormones. Chem Phys Lipids 189:1–6. CrossRefPubMedGoogle Scholar
  19. Ohanian J, Ohanian V (2001) Lipid second messenger regulation: the role of diacylglycerol kinases and their relevance to hypertension. J Hum Hypertens 15:93–98. CrossRefPubMedGoogle Scholar
  20. Pereyra CM, Ramella NA, Pereyra MA, Barassi CA, Creus CM (2010) Changes in cucumber hypocotyl cell wall dynamics caused by Azospirillum brasilense inoculation. Plant Physiol Biochem 48:62–69. CrossRefPubMedGoogle Scholar
  21. Peters C, Kim S-C, Devaiah S, Li M, Wang X (2014) Non-specific phospholipase C5 and diacylglycerol promote lateral root development under mild salt stress in Arabidopsis. Plant Cell Environ 37:2002–2013. CrossRefPubMedGoogle Scholar
  22. Pokotylo I, Pejchar P, Potocky M, Kocourkova D, Krckova Z, Ruelland E, Kravets V, Martinec J (2013) The plant non-specific phospholipase C gene family. Novel competitors in lipid signaling. Prog Lipid Res 52:62–79. CrossRefPubMedGoogle Scholar
  23. Pokotylo I, Kolesnikov Y, Kravets V, Zachowski A, Ruelland E (2014) Plant phosphoinositide-dependent phospholipases C: variations around a canonical theme. Biochimie 96:144–157. CrossRefPubMedGoogle Scholar
  24. Profotová B, Burketová L, Novotná Z, Martinec J, Valentová O (2006) Involvement of phospholipases C and D in early response to SAR and ISR inducers in Brassica napus plants. Plant Physiol Biochem 44:143–151. CrossRefPubMedGoogle Scholar
  25. Scherer GFE (1995) Activation of phospholipase A2 by auxin and mastoparan in hypocotyl segments from zucchini and sunflower. J Plant Physiol 145:483–490. CrossRefGoogle Scholar
  26. Singh A, Kanwar P, Pandey A, Tyagi AK, Sopory SK, Kapoor S, Pandey GK (2013) Comprehensive genomic analysis and expression profiling of phospholipase C gene family during abiotic stresses and development in rice. PLoS One 8:e62494. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Spaepen S, Dobbelaere S, Croonenborghs A, Vanderleyden J (2008) Effects of Azospirillum brasilense indole-3-acetic acid production on inoculated wheat plants. Plant Soil 312:15–23. CrossRefGoogle Scholar
  28. Steenhoudt O, Vanderleyden J (2000) Azospirillum, a free-living nitrogen-fixing bacterium closely associated with grasses: genetic, biochemical and ecological aspects. FEMS Microbiol Rev 24:487–506. CrossRefPubMedPubMedCentralGoogle Scholar
  29. Tsukagoshi H (2012) Defective root growth triggered by oxidative stress is controlled through the expression of cell cycle-related genes. Plant Sci 197:30–39. CrossRefPubMedGoogle Scholar
  30. Tuteja N, Sopory SK (2008) Plant signaling in stress: G-protein coupled receptors, heterotrimeric G-proteins and signal coupling via phospholipases. Plant Signal Behav 3:79–86CrossRefGoogle Scholar
  31. Vande Broek A, Lambrecht M, Eggermont K, Vanderleyden J (1999) Auxins upregulate expression of the indole-3-pyruvate decarboxylase gene in Azospirillum brasilense. J Bacteriol 181:1338–1342PubMedPubMedCentralGoogle Scholar
  32. Vermeer JEM, van Wijk R, Goedhart G, Geldner N, Chory J, Gadella TWJ Jr, Munnik T (2017) In vivo imaging of diacylglycerol at the cytoplasmic leaflet of plant membranes. Plant Cell Physiol 58:1196–1207. CrossRefPubMedPubMedCentralGoogle Scholar
  33. Vossen JH, Abd-El-Haliem A, Fradin EF, Van Den Berg G, Ekengren SK, Meijer HJG, Seifi A, Bai Y, Ten Have A, Munnik T, Thomma BPHJ, Joosten MHAJ (2010) Identification of tomato phosphatidylinositol-specific phospholipase-C (PI-PLC) family members and the role of PLC4 and PLC6 in HR and disease resistance. Plant J 62:224–239. CrossRefPubMedGoogle Scholar
  34. Wang X (2001) Plant phospholipases. Annu Rev Plant Physiol Plant Mol Biol 52:211–231. CrossRefPubMedGoogle Scholar
  35. Wang X (2004) Lipid signaling. Curr Opin Plant Biol 7:329–336. CrossRefPubMedGoogle Scholar
  36. Wimalasekera R, Pejchar P, Holk A, Martinec J, Scherer GF (2010) Plant phosphatidylcholine-hydrolyzing phospholipases C NPC3 and NPC4 with roles in root development and brassinolide signaling in Arabidopsis thaliana. Mol Plant 3:610–625. CrossRefPubMedGoogle Scholar

Copyright information

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2019

Authors and Affiliations

  1. 1.Instituto de Investigaciones Químico BiológicasUniversidad Michoacana de San Nicolás de Hidalgo, Ciudad UniversitariaMoreliaMexico

Personalised recommendations