A Complex Molecular Interplay of Auxin and Ethylene Signaling Pathways Is Involved in Arabidopsis Growth Promotion by Burkholderia phytofirmans PsJN

Abstract

Modulation of phytohormones homeostasis is one of the proposed mechanisms to explain plant growth promotion induced by beneficial rhizobacteria (PGPR). However, there is still limited knowledge about the molecular signals and pathways underlying these beneficial interactions. Even less is known concerning the interplay between phytohormones in plants inoculated with PGPR. Auxin and ethylene are crucial hormones in the control of plant growth and development, and recent studies report an important and complex crosstalk between them in the regulation of different plant developmental processes. The objective of this work was to study the role of both hormones in the growth promotion of Arabidopsis thaliana plants induced by the well-known PGPR Burkholderia phytofirmans PsJN. For this, the spatiotemporal expression patterns of several genes related to auxin biosynthesis, perception and response and ethylene biosynthesis were studied, finding that most of these genes showed specific transcriptional regulations after inoculation in roots and shoots. PsJN-growth promotion was not observed in Arabidopsis mutants with an impaired ethylene (ein2-1) or auxin (axr1-5) signaling. Even, PsJN did not promote growth in an ethylene overproducer (eto2), indicating that a fine regulation of both hormones signaling and homeostasis is necessary to induce growth of the aerial and root tissues. Auxin polar transport is also involved in growth promotion, since PsJN did not promote primary root growth in the pin2 mutant or under chemical inhibition of transport in wild type plants. Finally, a key role for ethylene biosynthesis was found in the PsJN-mediated increase in root hair number. These results not only give new insights of PGPR regulation of plant growth but also are also useful to understand key aspects of Arabidopsis growth control.

Keywords: AVG; EIN2; ETO2; IAA1; NPA; rhizobacteria; root development; root hairs.

FIGURE 1

Temporal quantitative real-time PCR of selected Arabidopsis genes involved in auxin synthesis and perception after inoculation with Burkholderia phytofirmans PsJN. Relative gene expression of selected genes involved auxin synthesis and perception in roots and shoots at 2, 24, 48, and 96 h post inoculation (hpi) with the strain. Data are means ± SE of 3–5 biological replicates per treatment, each considering tissue from 5 to 13 plants and two technical replicates. Normalization was performed with the housekeeping SAND family gene (AT2G28390). Asterisk indicates statistical significance among treatments in a particular time (One way ANOVA, p < 0.05).

FIGURE 2

Temporal quantitative real-time PCR of selected Arabidopsis genes involved in auxin transport and response after inoculation with B. phytofirmans PsJN. Relative gene expression of selected genes involved auxin transport and response in roots and shoots at 2, 24, 48, and 96 hpi with the strain. Data are means ± SE of 3–5 biological replicates per treatment, each considering tissue from 5 to 13 plants and two technical replicates. Normalization was performed with the housekeeping SAND family gene (AT2G28390). Asterisk indicates statistical significance among treatments in a particular time (One way ANOVA, p < 0.05).

FIGURE 3

Temporal quantitative real-time PCR of selected Arabidopsis genes related to ethylene biosynthesis after inoculation with B. phytofirmans PsJN. Relative gene expression of selected genes involved in the ethylene biosynthesis pathway in roots and shoots at 2, 24, 48, and 96 hpi with the strain. Data are means ± SE of 3–5 biological replicates per treatment, each considering tissue from 5 to 13 plants and two technical replicates. Normalization was performed with the housekeeping SAND family gene (AT2G28390). Asterisk indicates statistical significance among treatments in a particular time (One way ANOVA, p < 0.05).

FIGURE 4

Short-term effects of auxin transport inhibition and/or ethylene biosynthesis inhibition in Arabidopsis growth induction mediated by B. phytofirmans PsJN. Relative primary root length (A), rosette area (B), and fresh weight (C) in 14 days old plants inoculated (PsJN) or not (control) with strain PsJN. Plants were also grown without chemical inhibitors (MS), or under the chemical treatment with the auxin efflux inhibitor NPA (N-1-naphthylphthalamic acid) at 2 μM and/or with the ethylene biosynthesis inhibitor amino ethoxyvinyl glycine hydrochloride (AVG) at 1.5 μM. Data are means ± SE of at least 15 plants per treatment. Lowercase letters on top of the bars (a–e) indicate significant statistical differences among treatments. Bars that do not share an identical letter have significant differences, according to a Two-way ANOVA analysis (p < 0.05), considering inoculation and chemical treatments as factors, and a Tukey post hoc test (p < 0.05). Results are representative of three different experiments.

FIGURE 5

Effects of B. phytofirmans PsJN in root hair number and length under auxin transport and/or ethylene biosynthesis inhibition. Graphic representation of the root hair number (A) and hair length (B) of root tips (5 mm) from plants inoculated (PsJN) or not (control) with B. phytofirmans PsJN. Plants were also grown without chemical inhibitors (MS), or under the treatment with the auxin efflux inhibitor NPA (N-1-naphthylphthalamic acid) at 2 μM and/or with the ethylene biosynthesis inhibitor amino ethoxyvinyl glycine hydrochloride (AVG; Fluka Sigma–Aldrich) at 1.5 μM. Data are means ± SE of at least 20 plants per treatment. Lowercase letters on top of the bars (a–d) indicate significant statistical differences among treatments. Bars that do not share an identical letter have significant differences, according to a Two-way ANOVA analysis (p < 0.05), considering inoculation and chemical treatments as factors, and a Tukey post hoc test (p < 0.05). Results are representative of three different experiments. Representative photographs of each treatment are presented in (C); white bars in photographs correspond to 1 mm.

FIGURE 6

Effects of B. phytofirmans PsJN in different Arabidopsis mutants associated to auxin and ethylene pathways. Relative primary root length (A), rosette area (B) and fresh weight (C) in 14 days old wild type (wt) or mutant plants inoculated (PsJN) or not (control) with strain PsJN. Arabidopsis mutants lines used were eir1–1 (CS8058), iaa1 (CS16234), ein2-1 (CS3071), and eto2 (CS8059). Data are means ± SE of 17–35 plants per treatment, and represent at least four independent experiments. Asterisk indicates statistical significance between the inoculated and non-inoculated plants in each genotype (One way ANOVA, p < 0.05).