Plant Drought Tolerance Enhancement by Trehalose Production of Desiccation-Tolerant Microorganisms

Abstract

A collection of desiccation-tolerant xeroprotectant-producing microorganisms was screened for their ability to protect plants against drought, and their role as plant growth-promoting rhizobacteria was investigated in two different crops (tomato and pepper). The most commonly described biochemical mechanisms for plant protection against drought by microorganisms including the production of phytohormones, antioxidants and xeroprotectants were analyzed. In particular, the degree of plant protection against drought provided by these microorganisms was characterized. After studying the findings and comparing them with results of the closest taxonomic relatives at the species and strain levels, we propose that trehalose produced by these microorganisms is correlated with their ability to protect plants against drought. This proposal is based on the increased protection of plants against drought by the desiccation-sensitive microorganism Pseudomonas putida KT2440, which expresses the otsAB genes for trehalose biosynthesis in trans.

Keywords: Pseudomonas putida KT2440; desiccation tolerance; plant-growth-promoting rhizobacteria (PGPR); rhizobacterial drought-tolerance enhancers (RDTE); trehalose.

FIGURE 1

The effect of desiccation-tolerant microorganisms on pepper plants subjected to water stress. (A) Shows the physical appearance of pepper plants after 33 days without watering. (B,C) show the DW and FW (mg), respectively, of whole pepper plants free from soil. (D–G) show the FTW (mg), RWC, RL, and SL, respectively, of whole pepper plants free from soil. White bars correspond to day 7, light-gray bars correspond to day 14, dark bars correspond to day 21 and black bars correspond to day 33. Values are the means of three replicates ±SD.

FIGURE 2

Neighbor-joining phylogenetic tree based on the 16S rRNA sequence of strains Microbacterium sp. 3J1 and A. koreensis 5J12A (both with the latter’s closest relative strains), and A. piechaudii 366-5, used as outgroup. Numbers at the bifurcations indicate how many times each species occurred at this position. Bar, 0.01 changes per nucleotide position.

FIGURE 3

Antioxidant enzyme activity in different strains. (A) shows SOD in U_SOD/mL; and (B) shows CAT in U_CAT/mL (units of enzyme activity, U). Pseudomonas putida KT2440 was used as the control strain. Values are the means of three replicates ±SD.

FIGURE 4

Intracellular trehalose production in ppm/UFC (×10⁸)⋅mL^-1 by bacterial strains in different polyethylene glycol (PEG) concentrations. White bars correspond to cultures in the absence of PEG (0% PEG); gray bars, correspond to cultures supplemented with 5% PEG; and black bars, to cultures supplemented with 50% PEG. P. putida KT2440 was used as the control strain. Values are the means of three replicates ±SD.

FIGURE 5

Analysis of statistical correlation. One-way ANCOVA for the correlations between production values by each strain of trehalose, SOD, CAT, ACCd, IAA, GA₃, ABA, SA, and values for plant DW, FW, FTW, RWC, shoot length, and root length. Green circles correspond to Microbacterium sp. 3J1; red circles correspond to A. koreensis 5J12A; orange circles correspond to Rhodococcus sp. 4J2A2; violet circles correspond to Leucobacter sp. 4J7B1; blue circles correspond to A. siccitolerans 4J27; yellow circles correspond to P. putida KT2440, and gray circles correspond to non-inoculated samples.

FIGURE 6

Trehalose production by P. putida KT2440 containing the otsAB genes from Microbacterium sp. 3J1 and effect over pepper plants. (A) Shows the DW of pepper plants inoculated with P. putida KT2440 (pUCP22), P. putida (pUCP22:otsAB) and Microbacterium sp. 3J1. Where white bars correspond to day 0, light-gray bars correspond to day 7, black bars correspond to day 14. Values are the means of three replicates ±SD. (B) Shows the relative water content of pepper plants inoculated with P. putida KT2440 (pUCP22; triangles), P. putida (pUCP22:otsAB; squares), and Microbacterium sp. 3J1 (circles), as well as non-inoculated plants (diamonds) at time 0, 7, and 14 days. Values are the means of three replicates ±SD. (C) Shows the intracellular concentration of trehalose in the absence of PEG and in presence of 5 and 50% PEG. White bars correspond to cultures grown on M9 minimal medium, and gray bars to cultures grown on TSB medium. All bars show SD; ^abindicates a significant difference with respect to all other results; ^bindicates a significant difference with respect to control.