Nitric oxide synthase expression in Pseudomonas koreensis MME3 improves plant growth promotion traits

Abstract

The development of novel biotechnologies that promote a better use of N to optimize crop yield is a central goal for sustainable agriculture. Phytostimulation, biofertilization, and bioprotection through the use of bio-inputs are promising technologies for this purpose. In this study, the plant growth-promoting rhizobacteria Pseudomonas koreensis MME3 was genetically modified to express a nitric oxide synthase of Synechococcus SyNOS, an atypical enzyme with a globin domain that converts nitric oxide to nitrate. A cassette for constitutive expression of synos was introduced as a single insertion into the genome of P. koreensis MME3 using a miniTn7 system. The resulting recombinant strain MME3:SyNOS showed improved growth, motility, and biofilm formation. The impact of MME3:SyNOS inoculation on Brachypodium distachyon growth and N uptake and use efficiencies under different N availability situations was analyzed, in comparison to the control strain MME3:c. After 35 days of inoculation, plants treated with MME3:SyNOS had a higher root dry weight, both under semi-hydroponic and greenhouse conditions. At harvest, both MME3:SyNOS and MME3:c increased N uptake and use efficiency of plants grown under low N soil. Our results indicate that synos expression is a valid strategy to boost the phytostimulatory capacity of plant-associated bacteria and improve the adaptability of plants to N deficiency. KEY POINTS: • synos expression improves P. koreensis MME3 traits important for rhizospheric colonization • B. distachyon inoculated with MME3:SyNOS shows improved root growth • MME3 inoculation improves plant N uptake and use efficiencies in N-deficient soil.

Keywords: Brachypodium; Nitric oxide synthase; Nitrogen; Plant growth–promoting rhizobacteria; Pseudomonas.

Fig. 1

Genetic modification of P. koreensis MME3 to express synos. a Schematic representation of the miniTn7 carrying a synos expression cassette, inserted into the bacterial chromosome downstream of the glmS gene. GmR cassette confers resistance to gentamicin. The synos cassette is composed of the P_A1/04/03 promoter, the synos coding sequence, and the T_rrnB terminator. The half black arrows represent primers used to confirm the correct insertion of the miniTn7 (P₁ = Tn7GlmS and P₂ = GmR5ʹ RV) and the presence of synos (P₃ = SyNOSfw and P₄ = SyNOSrv). b Agarose gel electrophoresis of PCR products obtained by amplification with primers P₁ and P₂, showing the expected product of 442 bp, from two clones carrying the empty miniTn7(GmR) transposon (MME3:c1 and MME3:c2) as well as two clones carrying the synos miniTn7(GmR + SyNOS) transposon (MME3:SyNOS.1 and MME3:SyNOS.2). DNA ladder (100 bp) was included to determine product size (MW). c Agarose gel electrophoresis revealing partial PCR amplification of synos coding sequence with primers P₃ and P₄, showing the expected product of 1136 bp, from the recombinant clones. d Agarose gel electrophoresis of RT-PCR products obtained by partial amplification of synos transcript from MME3:SyNOS and MME3:c control strain. Total RNA extracts were used as template for RT-PCR using primers rtSyNOSfw and rtSyNOSrv (expected product of 178 bp)

Fig. 2

Growth kinetics of MME3:SyNOS and MME3:c (control) strains. Starter cultures grown in NB overnight were used to inoculate fresh NB + 5 mM l-Arg. MME3:c (gray circles) and MME3:SyNOS (black squares) were cultured under 150 rpm agitation at 28 °C. The OD at 600 nm was measured following the growth until reaching the stationary phase. The inset shows measurements done at the late stationary phase (48 h). For each condition, four independent replicas were used. Plotted values are means ± SE. Asterisks indicate statistically significant differences with a significance of p < 0.1 (*) or p < 0.05 (**) according to t-test analyses

Fig. 3

Phenotypic characterization of strain MME3:SyNOS. Bacterial cultures of MME3:SyNOS and MME3:c were grown overnight in NB medium. Aliquots of 10 μL of bacterial suspensions containing approximately 10⁹ CFU.mL⁻¹ were used in each assay. Swarming motility, measured from the area of colony growth on NA medium containing 0.5% (w/v) agar + 0.5% (w/v) glucose with or without 5 mM l-Arg (a). Swimming motility, measured in the same way, on NA medium with 0.3% (w/v) agar with or without 5 mM l-Arg (b). Siderophore production in King’s B with or without 5 mM l-Arg was measured from the orange halos, as revealed by the O-CAS assay. In all cases, the halos or colony areas were determined with ImageJ software (c). Production of IAA in NB medium containing 5 mM l-Arg with or without 5 mM l-Trp as estimated by the Salkowski method (d). In vitro biofilm formation in static cultures in NB or LB media, with or without 5 mM l-Arg. Approximately 10⁷ CFU.mL.⁻¹ of MME3:c and MME3:SyNOS was seeded in 96-well plates and cultured for 2 days at 28 °C under static conditions. Biofilm formation was determined by the crystal violet method measuring OD at 550 nm and normalizing data by the total growth (OD₅₅₀/OD₆₀₀) (e). The number of adhered cells was analyzed by measuring the fluorescence intensity (excitation 480 nm; emission 528 nm) of the cells present in biofilms formed by MME3:c:GFP and MME3:SyNOS:GFP (A.U., arbitrary fluorescence units) (f). Plotted values are the average of four independent replicas. Plotted values are means ± SE. **Statistically significant differences (t-test, p < 0.05)

Fig. 4

Effect of inoculation on Brachypodium distachyon with P. koreensis MME3:SyNOS grown under semi-hydroponics in sand. Seeds of B. distachyon were surface-sterilized and stratified. The plates were placed in a germination chamber at 20 °C with a 16/8-h light/dark photoperiod. Seven-day-old seedlings were transferred to 400 cm³ pots containing sterile sand and inoculated with 100 µL of MME3:c or MME3:SyNOS bacterial suspension. The plants were grown semi-hydroponically in a chamber under a photoperiod of 16 h of light at 25 °C. Non-inoculated seedlings (ni) were used as a control. Plants were watered once a week with ATS containing high nitrogen (HN-Hy, 9 mM KNO₃) (a, b) or low nitrogen (LN-Hy, 1 mM KNO₃) concentrations (c, d). After 35 days, plants were harvested and fresh weights (FW) and dry weights (DW) of the shoot and DW of root were measured. Plotted values are the average of 15 independent plants ± SE. Results were statistically analyzed by ANOVA and Sidak’s multiple comparison post-test. *Statistically significant differences (p < 0.1)

Fig. 5

Effect of inoculation on B. distachyon with P. koreensis MME3:SyNOS grown in a greenhouse under different soil conditions. Seeds of B. distachyon were surface-sterilized and stratified. Seedlings were transferred to 2-L pots containing high N soil (HN-S) or low N soil (LN-S) consisting of mixtures of soil:sand:perlite in 1:1:1 or 1:4:4 ratios, respectively, and inoculated with 100 µL of MME3:c or MME3:SyNOS bacterial suspension. Non-inoculated (ni) seedlings were used as a control. Plants were grown in a greenhouse in winter. a After 35 days, half of the plants were harvested and dry weights of the shoot and root parts were measured. Plotted values are the average of five replicas in each of three blocks ± SE. The other 15 plants were harvested when the spikes were mature to analyze grain yield (b), and total N content in shoots (c) and grains (d). Results were statistically analyzed by ANOVA and Turkey’s test. *Statistically significant differences (p < 0.1)

Fig. 6

Effect of inoculation with P. koreensis MME3:SyNOS on N uptake and use efficiencies of B. distachyon grown in a greenhouse under different soil conditions. The plants were growth on high N soil (HN-S) or low N soil (LN-S), consisting of a mixture of soil:sand:perlite in 1:1:1 or 1:4:4 ratios, respectively, and inoculated with 100 µL of MME3:c or MME3:SyNOS bacterial suspension. Seedlings non-inoculated (ni) were used as a control. The N indexes were calculated from N content measurements at harvest time, when the spikes were mature. a N uptake efficiency (NUpE) was calculated as N_grain / N_available, where N available is the sum of all N sources measured. b N use efficiency (NUE) was calculated as yield / N_available. Plotted values are the average of five replicas in each of three blocks ± SE. Results were statistically analyzed by one-way ANOVA for each condition and Tukey’s test. Different letters indicate significant differences (p < 0.05)