Applications of ancestral sequence reconstruction for understanding the evolution of plant specialized metabolism

Studies of enzymes in modern-day plants have documented the diversity of metabolic activities retained by species today but only provide limited insight into how those properties evolved. Ancestral sequence reconstruction (ASR) is an approach that provides statistical estimates of ancient plant enzyme sequences which can then be resurrected to test hypotheses about the evolution of catalytic activities and pathway assembly. Here, I review the insights that have been obtained using ASR to study plant metabolism and highlight important methodological aspects. Overall, studies of resurrected plant enzymes show that (i) exaptation is widespread such that even low or undetectable levels of ancestral activity with a substrate can later become the apparent primary activity of descendant enzymes, (ii) intramolecular epistasis may or may not limit evolutionary paths towards catalytic or substrate preference switches, and (iii) ancient pathway flux often differs from modern-day metabolic networks. These and other insights gained from ASR would not have been possible using only modern-day sequences. Future ASR studies characterizing entire ancestral metabolic networks as well as those that link ancient structures with enzymatic properties should continue to provide novel insights into how the chemical diversity of plants evolved. This article is part of the theme issue 'The evolution of plant metabolism'.