Gene expression in tobacco low-nicotine mutants

Area of Science:

• Plant molecular biology
• Biochemistry
• Genetics

Background:

• Nicotine is a major alkaloid in tobacco, influencing its commercial value and physiological effects.
• The genetic and molecular basis of nicotine biosynthesis regulation remains incompletely understood.

Purpose of the Study:

• To identify and characterize genes involved in the regulation of nicotine biosynthesis in tobacco.
• To elucidate the roles of the Nic1 and Nic2 genes in controlling nicotine levels and related metabolic pathways.

Main Methods:

• Differential gene expression analysis using subtraction hybridization to identify cDNAs with altered transcript levels in nic1 nic2 mutant tobacco roots.
• Gene cloning, expression in Escherichia coli, and enzymatic activity assays to determine the function of candidate genes (A411 and A622).
• Analysis of gene expression patterns under different physiological conditions (hormonal regulation, developmental cues) and in different mutant backgrounds.

Main Results:

• Two cDNAs, A411 and A622, were isolated, showing significantly lower transcript levels in mutant roots.
• A411 encodes a protein with putrescine N-methyltransferase activity (key enzyme in nicotine synthesis), while A622's function requires further investigation.
• Expression of both genes is predominantly in roots and is coordinately regulated by the nic mutations and environmental factors like floral removal and auxin.

Conclusions:

• Nic1 and Nic2 are likely regulatory genes controlling nicotine biosynthesis in tobacco.
• The identified genes, particularly A411, play crucial roles in the nicotine biosynthetic pathway.
• Environmental and developmental signals modulate nicotine production through the regulation of these key genes.