Polysomes from expanded tobacco leaves

Area of Science:

• Plant Molecular Biology
• Biochemistry
• Molecular Genetics

Background:

• Intact polysome isolation is crucial for studying gene expression in plants.
• Tobacco (Nicotiana tabacum L.) leaf polysome integrity is sensitive to developmental stage.
• Previous methods required specific buffers tailored to leaf age.

Purpose of the Study:

• To determine optimal conditions for isolating intact polysomes from tobacco leaves across different developmental stages.
• To develop a universal extraction medium for efficient polysome isolation.
• To assess the translational activity of isolated polysomes and their RNA components.

Main Methods:

• Extraction of polysomes from young, medium, and mature tobacco leaves using various buffer compositions.
• Inclusion of specific reagents like ethyleneglycol-bis-(β-amino-ethyl ether)-N,N'-tetracetic acid (EGTA), mercaptoethanol, and diethyldithiocarbamate (DIECA).
• Utilizing Nonidet-P40 (NP40) as a nonionic detergent in the extraction buffer.
• Assessing polysome translational activity using a wheat-germ in-vitro protein-synthesizing system.

Main Results:

• Polysome isolation efficiency and integrity varied significantly with leaf age and extraction buffer composition.
• A comprehensive buffer (25 mM EGTA, 0.01 M mercaptoethanol, 25 mM DIECA, 0.5% NP40) proved effective for all developmental stages.
• Isolated tobacco leaf polysomes demonstrated active protein synthesis in the wheat-germ system.
• Translational products were comparable using polysomes, polysomal RNA, or polysomal poly(A)(+) RNA, while poly(A)(-) RNA was ineffective.

Conclusions:

• A single, optimized extraction buffer enables reliable isolation of intact and translationally active polysomes from tobacco leaves of all ages.
• This method facilitates robust analysis of tobacco gene expression at the translational level.
• Polyadenylated RNA is essential for efficient in-vitro translation in the wheat-germ system.