Accelerated evolution and coevolution drove the evolutionary history of AGPase sub-units during angiosperm radiation

Abstract

Background and aims: ADP-glucose pyrophosphorylase (AGPase) is a key enzyme of starch biosynthesis. In the green plant lineage, it is composed of two large (LSU) and two small (SSU) sub-units encoded by paralogous genes, as a consequence of several rounds of duplication. First, our aim was to detect specific patterns of molecular evolution following duplication events and the divergence between monocotyledons and dicotyledons. Secondly, we investigated coevolution between amino acids both within and between sub-units.

Methods: A phylogeny of each AGPase sub-unit was built using all gymnosperm and angiosperm sequences available in databases. Accelerated evolution along specific branches was tested using the ratio of the non-synonymous to the synonymous substitution rate. Coevolution between amino acids was investigated taking into account compensatory changes between co-substitutions.

Key results: We showed that SSU paralogues evolved under high functional constraints during angiosperm radiation, with a significant level of coevolution between amino acids that participate in SSU major functions. In contrast, in the LSU paralogues, we identified residues under positive selection (1) following the first LSU duplication that gave rise to two paralogues mainly expressed in angiosperm source and sink tissues, respectively; and (2) following the emergence of grass-specific paralogues expressed in the endosperm. Finally, we found coevolution between residues that belong to the interaction domains of both sub-units.

Conclusions: Our results support the view that coevolution among amino acid residues, especially those lying in the interaction domain of each sub-unit, played an important role in AGPase evolution. First, within SSU, coevolution allowed compensating mutations in a highly constrained context. Secondly, the LSU paralogues probably acquired tissue-specific expression and regulatory properties via the coevolution between sub-unit interacting domains. Finally, the pattern we observed during LSU evolution is consistent with repeated sub-functionalization under 'Escape from Adaptive Conflict', a model rarely illustrated in the literature.

Fig. 1.

Maximum likelihood trees of angiosperm (A) small sub-unit (SSU) and (B) large sub-unit (LSU) sequences. Physcomitrella patens was used as the outgroup. Bootstrap values >60 % are indicated at each node. Branch lengths are proportional to the number of synonymous substitutions per synonymous site (dS estimated using PAML). Sequences are identified by species names followed by a number when several paralogue sequences were available for the same species. The tissue where expression occurred is indicated in parentheses when available (see Supplementary Data Tables S1 and S2). Branches tested for accelerated evolution are indicated by red upper case (target branches from A to G and from A to R in SSU and LSU, respectively) and lower case (control branches from a to h, and from a to i in SSU and LSU, respectively) letters. Target and control branch letters are also reported next to the name of the outgroup sequence used for each test. δ_n, node corresponding to a duplication event, with n = 1 to 2 or 5 for SSU or LSU, respectively. χ_n, nodes corresponding to the divergence between monocots and dicots, with n = 1 and n = 1–3 for SSU and LSU, respectively. *, sequences removed when testing accelerated evolution in branches A and B for SSU and branches C and D for LSU. +, sequences removed when testing accelerated evolution in branches Q and R for LSU.

Fig. 1.

Maximum likelihood trees of angiosperm (A) small sub-unit (SSU) and (B) large sub-unit (LSU) sequences. Physcomitrella patens was used as the outgroup. Bootstrap values >60 % are indicated at each node. Branch lengths are proportional to the number of synonymous substitutions per synonymous site (dS estimated using PAML). Sequences are identified by species names followed by a number when several paralogue sequences were available for the same species. The tissue where expression occurred is indicated in parentheses when available (see Supplementary Data Tables S1 and S2). Branches tested for accelerated evolution are indicated by red upper case (target branches from A to G and from A to R in SSU and LSU, respectively) and lower case (control branches from a to h, and from a to i in SSU and LSU, respectively) letters. Target and control branch letters are also reported next to the name of the outgroup sequence used for each test. δ_n, node corresponding to a duplication event, with n = 1 to 2 or 5 for SSU or LSU, respectively. χ_n, nodes corresponding to the divergence between monocots and dicots, with n = 1 and n = 1–3 for SSU and LSU, respectively. *, sequences removed when testing accelerated evolution in branches A and B for SSU and branches C and D for LSU. +, sequences removed when testing accelerated evolution in branches Q and R for LSU.

Fig. 2.

Position of residues exhibiting either accelerated evolution or coevolution. Positions are given along reference protein sequences of (A) the small (SSU) and (B) the large (LSU) sub-unit (accession nos AF330035 and S48563, respectively). Shaded boxes indicate motifs previously identified as being involved in protein activity, regulation or subunit interaction as follows. (A) 140–149, ATP-binding motif; 166–176, catalytic motif; 216–227, glucose-1-phosphate (G1P)-binding motif; 322–376, between-subunit interaction (BSI) motif; 458–the end, regulation motif. (B) 179–187, ATP-binding motif; 210–219, catalytic motif; 259–268, G1P-binding motif; 364–417, putative BSI motif, i.e. the region homologous to the SSU BSI domain; 499–the end, regulation motif. Letters above the sequence indicate the branch(es) in which sites were found to be under selection. We reported only sites with posterior probability (PP) (using the Bayes Empirical Bayes procedure) >0·90. Bold, sites with PP >0·95. Coevolving sites are denoted by a triangle for within-subunit and by an inverted triangle for between-subunit coevolution.

Fig. 3.

Pairs of coevolving residues in the AGPase small sub-unit. Position of the coevolving residues SSU-G360 and SSU-V461 (A), and SSU-F141 and SSU-G225 (B), are given on the small sub-unit 3-D structure diagram from Jin et al. (2005). Coloured segments indicate functionally known domains. Cat, catalytic motif; Reg, regulatory motif; G1P, glucose-1-phosphate interaction motif; ATP, ATP-binding motif; BSI, between-sub-unit interaction motif.