Abstract
Plant growth promoting rhizobacteria influence host functional and adaptive traits via complex mechanisms that are just started to be clarified. Azospirillum brasilense acts as a probiotic bacterium, but detailed information about its molecular mechanisms of phytostimulation is scarce. Three interaction systems were established to analyze the impact of A. brasilense Sp245 on the phenotype of Arabidopsis seedlings, and underlying molecular responses were assessed under the following growth conditions: (1) direct contact of roots with the bacterium, (2) chemical communication via diffusible compounds produced by the bacterium, (3) signaling via volatiles. A. brasilense Sp245 improved shoot and root biomass and lateral root production in the three interaction systems assayed. Cell division, quiescent center, and differentiation protein reporters pCYCB1;1::GUS, WOX5::GFP, and pAtEXP7::GUS had a variable expression in roots depending of the nature of interaction. pCYCB1;1::GUS and WOX5::GFP increased with volatile compounds, whereas pAtEXP7::GUS expression was enhanced towards the root tip in plants with direct contact with the bacterium. The auxin reporter DR5::GUS was highly expressed with diffusible and volatile compounds, and accordingly, auxin signaling mutants pin3, slr1, arf7arf19, and tir1afb2afb3 showed differential phytostimulant responses when compared with the wild type. By contrast, ethylene signaling was not determinant to mediate root changes in response to the different interactions, as observed using the ethylene-related mutants etr1, ein2, and ein3. Our data highlight the diverse effects by which A. brasilense Sp245 improves plant growth and root architectural traits and define a critical role of auxin but not ethylene in mediating root response to bacterization.
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Acknowledgments
This study was supported by the Coordinación de la Investigación Científica, Universidad Michoacana de San Nicolás de Hidalgo, México. We thank the Consejo Nacional de Ciencia y Tecnología (CONACYT) for the postgraduate scholarship to MMG (276783). We also sincerely thank León Francisco Ruiz-Herrera for technical assistance for confocal microscopy.
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MMG and EGP designed the experiments. MMG and SBO conducted the experiments. JLB and ECM provided materials, reagents, and technical support. MMG, EGP, and JLB analyzed the data and wrote the manuscript. All authors read and approved the manuscript.
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Supplementary Figure 1.
Characterization of the direct contact Azospirillum-Arabidopsis interaction. (a) Root images exposed to direct contact or diffusible compounds of the rhizobacterium. Bar = 200 μm. (b) Colonization analysis in roots. CFU = Colony Forming Units; C = Control (untreated roots); DC = Direct Contact; Dif = Diffusible compounds. n = 30. (PNG 347 kb)
Supplementary Figure 2.
Characterization of the interaction Azospirillum-Arabidopsis through the production of diffusible compounds. (a) Representative images of seedlings 6 d after transplant to medium containing A. brasilense (Ab), Autoclaved Ab (A-Ab), or E.coli. Bar = 1 cm. (b-f) Primary root length, lateral root number, lateral root density, shoot fresh weight and root fresh weight, respectively. (g) Representative images of root hair formation. Bar = 1 mm. (h,i) Root hair number and box-plot of root hair length, respectively. (j) Representative images of root hair formation after diffusible IAA treatment. Bar = 1 mm. (k,l) Root hair number and box-plot of root hair length, respectively. (m,n) Expression of the auxin response marker DR5::GUS. Bar = 100 μm. The rhizobacterium was used at 103 CFU/mL, and E. coli XL1 at 1.0 OD600. The letters on the bars indicate statistically significant differences according to Tukey's HSD test, P <0.05. (PNG kb)
Supplementary Figure 3.
Characterization of the interaction Azospirillum-Arabidopsis through the production of volatile compounds. (a) Representative images of seedlings 6 d after transplant to divided Petri dishes with medium containing A. brasilense (Ab), Autoclaved Ab (A-Ab), or E.coli. Bar = 1 cm. (b-f) Primary root length, lateral root number, lateral root density, shoot fresh weight and root fresh weight, respectively. (g) Representative images of root hair formation. Bar = 1 mm. (h,i) Root hair number and box-plot of root hair length, respectively. The rhizobacterium was used at 103 CFU/mL, and E. coli XL1 at 1.0 OD600. The letters on the bars indicate statistically significant differences according to Tukey's HSD test, P <0.05. (PNG 1.78 mb)
Supplementary Figure 4.
Effect of A. brasilense Sp245 on expression of the auxin response marker DR5::GUS. Representative images of the shoot, root and primary root meristem of seedlings 6 days after transplant with 103 CFU/mL of the rhizobacterium. As a positive control, plants were treated for 10 h with 0.05 μM of IAA in liquid MS medium. Bars in shoot and root = 1 mm; bar in root meristem = 100 μm. n = 30. The experiments were performed least three times with similar results. (PNG 1219 kb)
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Méndez-Gómez, M., Barrera-Ortiz, S., Castro-Mercado, E. et al. The nature of the interaction Azospirillum-Arabidopsis determine the molecular and morphological changes in root and plant growth promotion. Protoplasma 258, 179–189 (2021). https://doi.org/10.1007/s00709-020-01552-7
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DOI: https://doi.org/10.1007/s00709-020-01552-7