Desert-adapted plant growth-promoting pseudomonads modulate plant auxin homeostasis and mitigate salinity stress

Abstract

By providing adaptive advantages to plants, desert microorganisms are emerging as promising solutions to mitigate the negative and abrupt effects of climate change in agriculture. Among these, pseudomonads, commonly found in soil and in association with plants' root system, have been shown to enhance plant tolerance to salinity and drought, primarily affecting root system architecture in various hosts. However, a comprehensive understanding of how these bacteria affect plant responses at the cellular, physiological and molecular levels is still lacking. In this study, we investigated the effects of two Pseudomonas spp. strains, E102 and E141, which were previously isolated from date palm roots and have demonstrated efficacy in promoting drought tolerance in their hosts. These strains colonize plant roots, influencing root architecture by inhibiting primary root growth while promoting root hair elongation and lateral root formation. Strains E102 and E141 increased auxin levels in Arabidopsis, whereas this effect was diminished in IAA-defective mutant strains, which exhibited reduced IAA production. In all cases, the effectiveness of the bacteria relies on the functioning of the plant auxin response and transport machinery. Notably, such physiological and morphological changes provide an adaptive advantage to the plant, specifically under stress conditions such as salinity. Collectively, this study demonstrates that by leveraging the host's auxin signalling machinery, strains E102 and E141 significantly improve plant resilience to abiotic stresses, positioning them as potential biopromoters/bioprotectors for crop production and ecosystem restoration in alignment with Nature-based Solution approaches.

FIGURE 1

Root colonization patterns of endophytic bacteria E102 and E141 in Arabidopsis. Representative image of Arabidopsis Col‐0 root after 3 days after treatment (d.a.t.) with (A) water (mock, used as control), 10⁷ cell/mL of (B) E102 and (D) E141 GFP‐labelled strains. The GFP fluorescence of bacteria cells is visible in green, and plant tissues are visualized using red auto‐fluorescence. The root of control plants does not show any GFP fluorescence signals. Scale bars, 100 μm. Magnifications of the internal root tissues in (C) E102^Kan‐GFP and (E, F) E141^Kan‐GFP treated plants; white arrows indicate bacterial cells within the tissues. Root tissue of mock plants is reported as insert in panel C. Scale bar, 50 μm.

FIGURE 2

Effects of Pseudomonas spp. E102 and E141 treatments on root hair proliferation. (A) Representative images at 3 d.a.t. of Arabidopsis Col‐0 WT root exposed to water (mock) and 10⁷ bacterial cells/plate of E102 and E141 strains. Images are captured using both confocal and stereo microscopes. Scale bar, 200 μm. (B) Representative images at 3 d.a.t. of WT roots exposed to varying concentrations of E102 and E141 bacterial cells, that is, 10¹, 10³, 10⁵ and 10⁷ cells/plate. (C) The average number (n = 10–14) of root hairs above the root tip is reported for the different concentrations used for E102 (black dots) and E141 (grey dots). Values of bacterial cells/plate are expressed as log₁₀‐transformed numbers. Lowercase and capital letters indicate the results of the post hoc multiple comparisons of Tukey's tests (significance, p < 0.05) across the different bacteria concentrations used in E102 and E141, respectively. (D) Representative confocal images at 3 d.a.t. of Arabidopsis reporter line Col‐0 pWER::CFP exposed to water and 10⁷ bacterial cells/plate of E102 and E141 strains. The expression of cyan fluorescent protein (CFP) in N‐cells is indicated by the cyan colour. Roots were stained with propidium iodide to visualize the cell profile (red colour); scale bar, 100 μm.

FIGURE 3

Effects of Pseudomonas spp. E102 and E141 treatments on root architecture. (A) Representative images of 14 d.a.t. Arabidopsis Col‐0 plants treated with water (mock, 0 bacterial cells) and the maximum concentration of E102 and E141 (10⁷ bacterial cells/plates). (B) The average (n = 12–15) length of primary root (CM) in seedlings at 7 d.a.t. exposed to different concentrations of bacterial cells, that is, 10¹, 10³, 10⁵ and 10⁷ cells/plate, is shown for E102 (black dots) and E141 (grey dots). Values of bacterial cells/plate are expressed as log₁₀‐transformed numbers. Lowercase and capital letters indicate the results of the post hoc multiple comparisons of Tukey's tests (significance, p < 0.05) among the different bacteria concentrations used in E102 and E141, respectively. (C) Lateral root primordia reporter lines (pPLT3::GUS) are observed at 3 d.a.t. after GUS staining in mock, E102 and E141 roots; pPLT3::GUS expression was indicated by the blue colour. For each treatment, the scale bars are 50 μm and 100 μm in the left and right panels, respectively. (D) The average (n = 12–15) lateral root in seedlings at 7 d.a.t. exposed to different concentrations of bacterial cells in E102 (black dots) and E141 (grey dots). Values of bacterial cells/plate are expressed as log₁₀‐transformed numbers. Lowercase and capital letters indicate the results of the post hoc multiple comparisons of Tukey's tests (significance, p < 0.05) across the different bacteria concentrations used in E102 and E141, respectively.

FIGURE 4

Effects of Pseudomonas spp. E012 and E141 strains on auxin response in Arabidopsis. (A–H) Confocal images show the expression patterns of the auxin response genes DR5 using the Col‐0 reporter line DR5::GFP. Visualization at the confocal microscope of primary roots of DR5::GFP line at 3 d.a.t. with (A) water (mock), (B, C) 10⁷ bacterial cells/plate of E102 and E141 strains. The arrows indicate the expression of DR5::GFP along the root axis. (D) A representative image of DR5::GFP expression in plants exposed to 100 nM of indole acetic acid (IAA) 24 h after treatment is also reported. Scale bar 100 μm. (E–H) Arrows indicate DR5 expression under salinity stress (100 mM NaCl) in (F) absence and (G, H) presence of bacteria; coloured areas/cells indicated by stars represent the damages at the cellular level induced by salt. Mock non‐stressed is reported in panel E. (I–M) Representative images of Arabidopsis Col‐0 mutant axr3‐1 with reporter system DR5::GFP (axr3‐1 DR5::GFP) at 3 d.a.t.; this mutant exhibits auxin‐insensitive phenotypes. The expression of the green fluorescent protein (GFP) indicates the presence of auxin. Scale bar, 100 μm. (L, M) Treatments with E102 and E141 do not modify the auxin response of plants in axr3‐1, impairing the bacteria‐mediated phenotype observed in Arabidopsis Col‐0 (i.e. root hair formation in B, C). Yet, in axr3‐1 mutant, the two bacteria cannot protect plants from the effect of salinity (cellular damage in the inserts of L and M panels). (N–P) Arabidopsis Col‐0 treated with Yacasin and L‐Kyr (YL‐Kyr) to inhibit plant IAA production. (N) Mock plants show a reduced response of DR5 promoter, while the treatments with (O) E102 and (P) E141 increase the levels of DR5::GFP expression but fail to induce root hairs. Scale bar 100 μm.

FIGURE 5

Bacterial‐mediated modification of auxin transport in Arabidopsis. (A, B) Confocal images of transgenic Arabidopsis root expressing pAUX1::AUX1::YFP (reported as green) in mock and plants treated with E102 at 3 d.a.t., respectively. The white arrows point to increased fluorescence of the auxin influx transporter AUX1 in the differentiation zone. Scale bar 100 μm. (C, D) Confocal images of transgenic Arabidopsis root expressing pPIN2::PIN2::GFP (green) in mock and plants treated with E102 at 3 d.a.t., respectively. In all the images, roots were stained with propidium iodide to visualize the cell wall (red colour). (E–H) Confocal image showing the localization of pPIN2::PIN2::GFP in plants treated with (E) water (mock) and (F) E102; scale bar, 20 μm (G, H) Staining with the endocytic tracer FM46‐4 in mock and bacteria‐treated roots, respectively, indicates PIN2 internalisation and partial colocalization with the dye (see arrows indicating GFP green signal only vs. circles indicating GFP green and FM46‐4 red signals overlapped); scale bar, 5 μm. Arabidopsis mutant line (I–K) pin2 DR5::GUS and (L–N) aux1‐7 DR5::GUS in which the auxin efflux PIN2 and influx AUX1 transporters were defective, respectively. Activation of the DR5 promoter in Arabidopsis WT line for mock, E102 and E141 is reported as inserts.

FIGURE 6

Effects of Pseudomonas spp. E102 and E141 treatments on plant biomass. Fresh biomass (g) of (A) shoot and (B) root of Arabidopsis plantlets at 14 d.a.t. with water (mock) and different concentrations (10¹, 10³, 10⁵ and 10⁷ cells/plate) of E102 and E141 strains under three conditions: No stress (0 mM of NaCl), mild stress (50 mM of NaCl) and severe stress (100 mM of NaCl). Data were expressed as mean ± standard error of seedling' shoot and root (n = 7, from three replicates). Lower and capital case letters indicate the results of the post hoc multiple comparisons Tukey's tests (significance, p < 0.05) performed for E102 and E141 data sets, respectively, at each of the three conditions. Results were confirmed by three independent experiments. (C) Propidium iodide (PI) is used to investigate the damages induced by severe salt stress (100 mM NaCl) at the cellular level; representative confocal microscope images are reported for mock, E102 and E141 treatments (scale bar 100 μm).

FIGURE 7

A mechanistic model of the PGP process induced by Pseudomonas strains E102 and E141. Schematic illustration of the root colonization and modifications induced by E102 and E141 in Arabidopsis. Bacteria colonize the roots of plants, activating a series of modifications in the root system architecture by promoting root hair proliferation. These morphological changes are accompanied by alterations in PIN2 localization, as PIN2 protein becomes more internalized in vesicles alongside increased levels of the auxin response signal, as shown by the synthetic auxin‐responsive promoter DR5. Modifications induced by the bacterial treatment provide an adaptive advantage to the plant under severe salinity stress (100 mM NaCl), as treated plants exhibited greater biomass and showed no cellular damage caused by salinity.