Improving the plant-growth promoting ability of Azospirillum brasilense by genetic manipulation

Abstract

Certain plant growth promoting bacteria, by hydrolyzing 1-aminocyclopropane-1-carboxylic acid, ACC (the precursor of ethylene), through the action of ACC deaminase, regulate the production of ethylene in plants, promoting root elongation and decreasing the deleterious effects caused by stress ethylene. Azospirillum, a PGPB that promotes the growth of numerous plant species, does not produce ACC deaminase.

A. brasilense Cd transformed with the ACC deaminase gene (acdS) from Enterobacter cloacae UW4 did not show any ACC deaminase activity. To overcome the lack of expression of acdS in Azospirillum, the wild type promoter of acdS was replaced with the lac promoter, cloned in the broad host range plasmid pRK415, and transferred into Azospirillum brasilense Cd and A. brasilense Sp245.

The roots of tomato and canola seedlings were significantly longer in plants inoculated with A. brasilense Cd/pRKLACC transformants (acdS under the control of the lac promoter) than those in plants inoculated with the non-transformed strain. In the case of wheat seedlings, inoculation with A. brasilense Cd transformants did not promote root growth. The difference in plant response (canole and tomato versus wheat) is attributed to the higher sensitivity to ethylene of canola and tomato plants as compared to wheat plants.

Inferring that the promoter of a tetracycline resistance gene (Tet) might impose less metabolic load on A. brasilense Cd cells than the lac promoter, acdS was fused to the Tet gene promoter by PCR, cloned in pRK415, and transferred into A. brasilense Cd.

A. brasilense Cd/pRKLACC transformants showed higher ACC deaminase activity than the Cd/pRKTACC transformants (acdS under the control of the Tet promoter). However, the Cd/pRKLACC transformants showed a decrease in IAA synthesis, a lower growth rate, and a decreased ability to survive on the surface of tomato leaves as compared to the Cd/pRKTACC transformants.

Fresh and dry shoot weight of flooded tomato plants (Lycopersicum esculentun Mill. cv. Heinz 1439 VF) inoculated with A. brasilense Cd/pRKTACC was higher than plants inoculated with A. brasilense Cd/pRKLACC or non-transformed A. brasilense Cd. The level of epinasty of the same plants inoculated with A. brasilense Cd/pRKTACC was lower than in plants inoculated with A. brasilense Cd/pRKLACC was lower than in plants inoculated with A. brasilense Cd/pRKLACC or non-transformed A. brasilense Cd. Experiments on tomato plants exposed to Fusarium oxysporum, Pythium aphanidermatum, or Pseudomonas syringae and inoculated with A. brasilense Cd/pRKTACC or non-transformed A. brasilense Cd suggested that both the transformed and the non-transformed strain have biocontrol properties against the phytopathogens. The better protection provided by A. brasilense Cd/pRKTACC (when inoculated on tomato leaves) against pathogen attack suggest that the ability of the transformants to break down ACC deaminase lowers the levels of ethylene evolved by the plant as a response to pathogen attack. Further experiments are required to confirm this hypothesis.

The possibility of using A. brasilense Cd/pRKLACC in mangrove reforestation programs is discussed.