Maize drought protection by Azospirillum argentinense Az19 requires bacterial trehalose accumulation

Abstract

Azospirillum argentinense Az19 is an osmotolerant plant growth-promoting bacterium that protects maize plants from drought. In this work, we explored the role of trehalose in the superior performance of Az19 under stress. The trehalase-coding gene treF was constitutively expressed in Az19 through a miniTn7 system. The resulting recombinant strain, Az19F, did not accumulate trehalose, was affected in its capacity to cope with salt-, osmotic-, and UV-stress, and showed higher reactive oxygen species levels. Physiological alterations were also observed under normal conditions, such as increased growth in biofilms, higher motility, and decreased auxin secretion. Even so, the capacity of Az19F to colonize maize roots was not affected, either under normal or drought conditions. When inoculated in maize, both Az19 and Az19F strains promoted plant growth similarly under normal irrigation. However, unlike Az19, the trehalose-deficient strain Az19F could not improve the height, aerial fresh weight, or relative water content of maize plants under drought. Notably, Az19F triggered an exacerbated oxidative response in the plants, resulting in higher levels of antioxidant and phenolic compounds. We conclude that the role of trehalose metabolism in A. argentinense Az19 transcends stress tolerance, being also important for normal bacterial physiology and its plant growth-promoting activity under drought. KEY POINTS: • Trehalose is required by Az19 for full tolerance to salt-, osmotic-, and UV-stress. • A restriction in trehalose accumulation alters Az19 normal cell physiology. • Trehalose contributes to Az19-induced maize growth promotion under drought.

Keywords: Abiotic stress; Biofertilizer; Inoculant; Osmolyte; PGPR.

Fig. 1

Construction of the trehalose-depleted recombinant strain A. argentinense Az19F. A Schematic representation of the constructed miniTn7, carrying gentamicin resistance (Gm^r) and treF-expression cassettes. B PCR amplification products confirming the correct insertion of the miniTn7 in the chromosome of the recombinant clones C1, C2 and C3, and its absence in Az19 (wt). C Genomic region downstream of glmS gene, where the miniTn7 was inserted (black arrows) D Screening of conditions for NaCl-induced trehalose accumulation in A. argentinense Az19 by TLC. Three NaCl concentrations (0, 150 and 300 mM) and two induction times (24 and 48 h) were tested. A trehalose standard was used as reference (Tre). E Confirmation of trehalose degradation in the mutant strain Az19F by TLC. Glucose (Glu), sucrose (Suc), maltose (Mal) and trehalose (Tre) standards were used as references

Fig. 2

Effect of trehalose degradation on Az19 stress tolerance. A Salinity tolerance was evaluated as the bacterial survival after 48 h of incubation in NYA medium containing 200 mM NaCl. Survival was estimated as the percentual relation between the final and initial CFU number. B Representative photographs of 35 mm Petri dishes containing bacterial cultures after incubation in normal conditions or salt stress. C Tolerance to 20% PEG after 48 h of growth in NYA medium. D Tolerance to UV stress was estimated as the percentage of surviving CFU that grew on RC plates after irradiating with UVC light, in comparison to non-treated plates. E Representative photographs of Az19gm and Az19F colonies formed after UV treatment. F Percentage of surviving cells after receiving a heat-shock at 50 °C for 30 or 45 min. G Percentage of surviving cells after being frozen at −20 °C for 5 or 15 days. H Quantification of Az19gm and Az19F intracellular ROS levels under control or salt-stress conditions, using the probe H₂DCFDA. Asterisks denote significant differences according to a paired t-test. Different letters in (H) denote significant differences according to two-way ANOVA plus Tukey’s test (p ≤ 0.05)

Fig. 3

Effect of trehalose degradation on Az19 physiological characteristics associated with plant growth-promotion. Characterization of Az19gm and Az19F PGP-related traits under salt stress (300 mM NaCl) or without it (Control). A, B, and C Strains Az19gm and Az19F were cultured statically in NYA medium to analyze biofilm production (A), planktonic and adherent growth (B), and EPS production (C). D Swimming mobility of Az19gm and Az19F in semisolid NYA medium. E Auxinic compounds secreted by Az19gm and A19F after 48 h of growth in NYA + tryptophan. F Az19 and Az19F recovery from maize seeds 2 h after inoculation (2 hpi), or from the roots after 7 days of growth (7 dpi) under normal irrigation or drought. In all cases, different letters indicate statistically significant differences according to two-way ANOVA plus Tukey’s multiple comparison test (p ≤ 0.05)

Fig. 4

Effect of trehalose degradation on Az19-induced maize drought tolerance. Maize seeds were inoculated with Az19 or Az19F, or not inoculated (Ni), sown in pots and incubated in a growth chamber under full (Control) or 50% field capacity (Drought). After 15 days, maize growth was analyzed by measuring plant height (A), fresh and dry weight of the shoot, dry weight of the root (B) and the leaf relative water content of stressed plants (C). Plots depict average values plus the standard deviation of three independent experiments. Data was analyzed by two-way ANOVA, plus Tukey’s multiple comparison test (p ≤ 0.05)

Fig. 5

Effect of Az19 trehalose degradation on the oxidative status of inoculated maize. Maize seeds were inoculated as described before, and the last completely expanded leaf of each plant was cut, immediately feezed in N₂ and kept at −80 °C until lyophilization. Samples were then processed to analyze total phenolic compounds (A) and antioxidant capacity by the ABTS and FRAP methods (B). Data was analyzed by two-way ANOVA, plus Tukey’s multiple comparison test (p ≤ 0.05)