Endophytic Bacillus and Pseudomonas spp. Modulate Apple Shoot Growth, Cellular Redox Balance, and Protein Expression Under in Vitro Conditions

Abstract

Interactions between host plants and endophytic microorganisms play an important role in plant responses to pathogens and environmental stresses and have potential applications for plant stress management under in vitro conditions. We assessed the effect of endophytic bacteria on the growth and proliferation of domestic apple cv. Gala shoots in vitro. Further, a model apple cell suspension system was used to examine molecular events and protein expression patterns at an early stage of plant-endophyte interaction. Among the seven strains used in the study, Bacillus spp. strains Da_1, Da_4, and Da_5 and the Pseudomonas fluorescens strain Ga_1 promoted shoot growth and auxiliary shoot proliferation. In contrast, Bacillus sp. strain Oa_4, P. fluorescens strain Ga_3 and P. orientalis strain G_12 inhibited shoot development. In the cell suspension, the effects of the association between endophytic bacteria and plant cells were specific to each strain. Modulation of the cellular redox balance was monitored in the apple cells using a 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA) probe, and strain-specific effects were observed that correlated with the in vitro shoot development results. Proteomic analysis revealed differences in protein expressions in apple cells co-cultivated with different Bacillus spp. strains that had contrasting effects on cellular redox balance and shoot development. The Bacillus sp. strain Da_4, which enhanced shoot development and oxidation of H₂DCFDA, induced differential expression of proteins that are mainly involved in the defense response and regulation of oxidative stress. Meanwhile, treatment with Bacillus sp. strain Oa_4 led to strong upregulation of PLAT1, HSC70-1 and several other proteins involved in protein metabolism and cell development. Taken together, the results suggest that different cell signaling and response events at the early stage of the plant-endophyte interaction may be important for strain-dependent regulation of cellular redox balance and development of shoot phenotype.

Keywords: micropropagation; plant stress; plant–endophyte interaction; proteomics; reactive oxygen and nitrogen species.

FIGURE 1

Combined weight of parent and auxiliary apple shoots (left panel) and the auxiliary shoot propagation coefficient (middle panel). Apple shoots were inoculated with 3 μL of each strain suspended at ∼10⁷ cfu/ml in Murashige-Skoog medium. After 3 weeks of co-cultivation, the combined weight of parent and auxiliary shoots and the propagation coefficient represented by an average number of auxiliary shoots that developed on the parent shoot were assessed. A dashed line indicates the mean value of the control shoots. The insert on the right shows a representative sample of auxiliary shoots derived from one parent shoot. Data are presented as mean and SEM of a total of 40–55 shoots from three independent experiments. Means followed by the same letter are not significantly different (p < 0.05).

FIGURE 2

Accumulation of MDA in the leaves of apple shoots co-cultivated with endophytic bacteria. Apple shoots were inoculated with 3 μL of each strain suspended at ∼10⁷ cfu/ml in (MS) medium and observations were made after 1 week. Data are presented as the mean and SEM of at least three repeats from three independent experiments. Means followed by the same letter are not significantly different (p < 0.05).

FIGURE 3

Association of endophytic bacteria with apple cells in suspension. Apple cell suspension was inoculated with each strain at a final concentration of ∼10⁷ cfu/ml. After 6 h of co-incubation, the apple cells were sedimented by centrifugation and associated bacterial cells were quantified by serial dilution. Data are presented as the mean and SEM of at least three repeats from three independent experiments. Means followed by the same letter are not significantly different (p < 0.05).

FIGURE 4

Effect of endophytic bacteria on the accumulation of ROS/RNS in apple cells. Apple cell suspension was inoculated with each strain at the final concentration of ∼10⁷ cfu/ml and observations was made after 2 and 6 h. Data are presented as mean and SEM of at least three repeats from three independent experiments. Means followed by the same letter are not significantly different (p < 0.05).

FIGURE 5

Hierarchical cluster analysis results of the abundance data of proteoforms differentially expressed in apple cells co-incubated with Bacillus spp. strains Da_4 or Oa_4 for 6 h. Numbers on the left indicate four major clusters based on expression patterns. Colors indicate a decrease (green) or increase (red) in protein abundance compared to control. Letters in columns 1–3 indicate statistically significant (p < 0.01) differences between the Da_4 treatment and control (a), Oa_4 treatment and control (b), and between the Da_4 and Oa_4 treatments (c). Protein names and symbols are shown in columns 3 and 4, respectively.

FIGURE 6

A protein interaction network using A. thaliana proteins most closely related to the proteins (groups 1, 2, and 4) that were differentially expressed in apple cells co-incubated with Bacillus spp. strains Da_4 or Oa_4. The protein interaction network was built using the String database. Circle colors correspond to the protein group: 1 – red, 2 – orange, 4 – green, and gray circles represent additional network hubs that were not identified in this study. Proteins related by similar functions are outlined with colored shapes.