The long road of functional recruitment—The evolution of a gene duplicate to pyrrolizidine alkaloid biosynthesis in the morning glories (Convolvulaceae)

Abstract

In plants, homospermidine synthase (HSS) is a pathway-specific enzyme initiating the biosynthesis of pyrrolizidine alkaloids (PAs), which function as a chemical defense against herbivores. In PA-producing Convolvulaceae ("morning glories"), HSS originated from deoxyhypusine synthase at least >50 to 75 million years ago via a gene duplication event and subsequent functional diversification. To study the recruitment of this ancient gene duplicate to PA biosynthesis, the presence of putative hss gene copies in 11 Convolvulaceae species was analyzed. Additionally, various plant parts from seven of these species were screened for the presence of PAs.Although all of these species possess a putative hss copy, PAs could only be detected in roots of Ipomoea neei (Spreng.) O'Donell and Distimake quinquefolius (L.) A.R.Simões & Staples in this study. A precursor of PAs was detected in roots of Ipomoea alba L. Thus, despite sharing high sequence identities, the presence of an hss gene copy does not correlate with PA accumulation in particular species of Convolvulaceae. In vitro activity assays of the encoded enzymes revealed a broad spectrum of enzyme activity, further emphasizing a functional diversity of the hss gene copies. A recently identified HSS specific amino acid motif seems to be important for the loss of the ancestral protein function-the activation of the eukaryotic initiation factor 5A (eIF5A). Thus, the motif might be indicative for a change of function but allows not to predict the new function. This emphasizes the challenges in annotating functions for duplicates, even for duplicates from closely related species.

In defense of Apocynaceae: inference on evolution of pyrrolizidine alkaloids from evolution of an enzyme in their biosynthetic pathway, homospermidine synthase

“…activation of eIF5A), rather than HSS function, via re-recruitment as a DHS. As in Apocynaceae, only a minority of species in Convolvulaceae with HSS-like genes produce PAs (Prakashrao, et al 2022). Also similar to the results here, the IXXXN HSS in Ipomoea alba (Convolvulaceae), a non-PA producing species within a genus that contains PA-producing species, is able to produce deoxyhypusine, unlike Convolvulaceae species with either IXXXD or VXXXD HSS (Prakashrao, et al 2022).…”

“…Five of the amino acids of particular interest (positions 74, 75, 246, 269, 273, identified via selection and/or functional assays) are linked to functional areas in human DHS. Positions 246, 269, and 273 are predicted to be near the enzyme surface at the active site tunnel entrance, so binding substrates likely encounter these amino acids (Prakashrao, et al 2022). Umland, et al (2004 showed that the human DHS tunnel entrance had a less negative charge than the modelled Senecio vulgaris (Asteraceae) HSS, leading to the prediction that certain amino acid substitutions in this area could affect binding affinity.…”

“…DHS-like function), but allow the entrance of much smaller polyamines (spermidine and putrescine; i.e. HSS-like function) (Umland, et al 2004;Ober and Kaltenegger 2009;Wator, et al 2020;Prakashrao, et al 2022). After experimental validation of the effect of these substitutions on function, a combination of these sites may be more effective at identifying functionally optimized HSS.…”

“…Abolition of DHS function would free a paralog to evolve elevated expression levels. However, Ipomoea alba (Convolvulaceae) expresses in vivo an IXXXN HSS with detectable DHS function in vitro (Prakashrao et al, 2022), suggesting that some DHS activity by HSS enzymes is tolerated. Does the N273D substitutional also increase HSS activity?…”

“…mutagenesis of Ipomoea alba IXXXN HSS to IXXXD only increased homospermidine production by ca. 20% (Prakashrao et al, 2022).…”