**2. To be or not to be hot: capsaicinoid presence as a genetic trait**

Some peppers are pungent, and others are not. Pepper pungency is a trait conferred by the presence of the functional allele of *Pun1* gene [4]. It is considered to be a dominant trait [5]. Still, the recent report of hot pepper hybrids derived from the crossing of two non-pungent parents [6] makes such a heterosis effect on capsaicinoid accumulation deserve further studies in the future.

The *Pun1* gene (also known as AT3) has been demonstrated to be involved in capsaicinoid biosynthesis. Firstly, there is a correlation between its expression with capsaicinoid accumulation and the lack of the pungent trait in pepper lines with mutations in this gene [4, 7]. Moreover, in the last years, several authors have demonstrated that virus silencing (VIGS) of the gene in pepper leads to a decrease in capsaicinoid accumulation [4, 8–10]. It should be noted that the pepper plant is recalcitrant to Agrobacterium transformation [3, 10]; therefore, the usual functional approach by genetic transformation of the plant has not been possible so far.

Another approach was to demonstrate that the protein encoded by Pun1/AT3 is the so-called capsaicin synthase. Ogawa et al. [9] expressed Pun1 protein in *Escherichia coli*, purified it, and obtained antibodies anti-Pun1, which inhibited capsaicinoid formation in an assay using pepper protoplasts. However, they did not assay the enzymatic activity of purified Pun1 itself. Recently, Muratovska et al. [11] expressed Pun1/AT3 and another aminotransferase gene (*CaAT*) in *Saccharomyces cerevisiae* and proved that both lines of transformed yeasts were able to synthesize the capsaicinoid nonivamide. Finally, Milde et al. [12] not only purified Pun1 expressed in *E. coli* but also demonstrated the ability of the purified protein to catalyze the capsaicin synthase reaction, that is, the transformation of trans 8-methyl-6-nonenoyl-CoA and vanilloylamine into capsaicin. Thus, Pun1 is a bonafide capsaicin synthase. However, the possibility that other pepper proteins, such as CaAT, could also be involved in the biocatalysis of this reaction cannot be excluded [11].
