Mikimopine synthase (mis) gene on pRi1724

Area of Science:

• Plant molecular biology
• Microbial genetics
• Biochemistry

Background:

• Agrobacterium rhizogenes harbors Ri plasmids containing T-DNA, which can modify plant genomes.
• Opines are compounds produced by plants in response to Agrobacterium infection, serving various roles.
• Mikimopine and cucumopine are distinct opines with stereoisomeric properties.

Purpose of the Study:

• To identify and characterize the gene responsible for mikimopine synthesis.
• To compare mikimopine synthase with cucumopine synthase.
• To functionally validate the identified gene in transgenic plants.

Main Methods:

• Nucleotide sequencing of T-DNA from pRi1724 Ri plasmid.
• Northern blotting to detect gene transcripts in transgenic hairy roots.
• Agrobacterium-binary vector system for introducing the gene into tobacco.
• Bacterial protein expression system (E. coli) for enzyme production and in vitro assays.
• Paper electrophoresis, TLC, and HPLC for opine identification.

Main Results:

• A novel open reading frame (ORF) encoding 318 amino acids was identified adjacent to the T-DNA right border of pRi1724.
• A 1.35 kb transcript from this ORF was detected in transgenic Ajuga reptans hairy roots.
• Transgenic tobacco plants expressing the ORF accumulated mikimopine in all organs.
• Mikimopine synthase and cucumopine synthase showed no nucleotide homology but partial amino acid homology.
• Both enzymes, produced in E. coli, synthesized their respective opines from L-histidine and alpha-ketoglutaric acid using NADH.

Conclusions:

• The identified ORF is the mikimopine synthase gene responsible for mikimopine production.
• Mikimopine synthase shares functional and partial structural similarities with cucumopine synthase.
• The study provides a functional characterization of a novel opine synthase gene and its product.