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Limited genome resources are a bottleneck in the study of horizontal transfer (HT) of DNA in plants. To solve this issue, we tested the usefulness of low-depth sequencing data generated from 19 previously uncharacterized panicoid grasses for HT investigation. We initially searched for horizontally transferred LTR-retrotransposons by comparing the 19 sample sequences to 115 angiosperm genome sequences. Frequent HTs of LTR-retrotransposons were identified solely between panicoids and rice (Oryza sativa). We consequently focused on additional Oryza species and conducted a nontargeted investigation of HT involving the panicoid genus Echinochloa, which showed the most HTs in the first set of analyses. The comparison of nine Echinochloa samples and ten Oryza species identified recurrent HTs of diverse transposable element (TE) types at different points in Oryza history, but no confirmed cases of HT for sequences other than TEs. One case of HT was observed from one Echinochloa species into one Oryza species with overlapping geographic distributions. Variation among species and data sets highlights difficulties in identifying all HT, but our investigations showed that sample sequence analyses can reveal the importance of HT for the diversification of the TE repertoire of plants.

Novel adaptations are generally assembled by co-opting pre-existing genetic components, but the factors dictating the suitability of genes for new functions remain poorly known. In this work, we used comparative transcriptomics to determine the attributes that increased the likelihood of some genes being co-opted for C4 photosynthesis, a convergent complex trait that boosts productivity in tropical conditions. We show that independent lineages of grasses repeatedly co-opted the gene lineages that were the most highly expressed in non-C4 ancestors to produce their C4 pathway. Although ancestral abundance in leaves explains which genes were used for the emergence of a C4 pathway, the tissue specificity has surprisingly no effect. Our results suggest that levels of key genes were elevated during the early diversification of grasses and subsequently repeatedly used to trigger a weak C4 cycle via relatively few mutations. The abundance of C4-suitable transcripts therefore facilitated physiological innovation, but the transition to a strong C4 pathway still involved consequent changes in expression levels, leaf specificity, and coding sequences. The direction and amount of changes required for the strong C4 pathway depended on the identity of the genes co-opted, so that ancestral gene expression both facilitates adaptive transitions and constrains subsequent evolutionary trajectories.

Rubisco is responsible for the fixation of CO 2 into organic compounds through photosynthesis and thus has a great agronomic importance. It is well established that this enzyme suffers from a slow catalysis, and its low specificity results into photorespiration, which is considered as an energy waste for the plant. However, natural variations exist, and some Rubisco lineages, such as in C 4 plants, exhibit higher catalytic efficiencies coupled to lower specificities. These C 4 kinetics could have evolved as an adaptation to the higher CO 2 concentration present in C 4 photosynthetic cells. In this study, using phylogenetic analyses on a large data set of C 3 and C 4 monocots, we showed that the rbcL gene, which encodes the large subunit of Rubisco, evolved under positive selection in independent C 4 lineages. This confirms that selective pressures on Rubisco have been switched in C 4 plants by the high CO 2 environment prevailing in their photosynthetic cells. Eight rbcL codons evolving under positive selection in C 4 clades were involved in parallel changes among the 23 independent monocot C 4 lineages included in this study. These amino acids are potentially responsible for the C 4 kinetics, and their identification opens new roads for human-directed Rubisco engineering. The introgression of C 4 -like high-efficiency Rubisco would strongly enhance C 3 crop yields in the future CO 2 -enriched atmosphere.

The evolutionary accessibility of novel adaptations varies among lineages, depending in part on the genetic elements present in each group. However, the factors determining the evolutionary potential of closely related genes remain largely unknown. In plants, CO2-concentrating mechanisms such as C4 and crassulacean acid metabolism (CAM) photosynthesis have evolved numerous times in distantly related groups of species, and constitute excellent systems to study constraints and enablers of evolution. It has been previously shown for multiple proteins that grasses preferentially co-opted the same gene lineage for C4 photosynthesis, when multiple copies were present. In this work, we use comparative transcriptomics to show that this bias also exists within Caryophyllales, a distantly related group with multiple C4 origins. However, the bias is not the same as in grasses and, when all angiosperms are considered jointly, the number of distinct gene lineages co-opted is not smaller than that expected by chance. These results show that most gene lineages present in the common ancestor of monocots and eudicots produced gene descendants that were recruited into C4 photosynthesis, but that C4-suitability changed during the diversification of angiosperms. When selective pressures drove C4 evolution, some copies were preferentially co-opted, probably because they already possessed C4-like expression patterns. However, the identity of these C4-suitable genes varies among clades of angiosperms, and C4 phenotypes in distant angiosperm groups thus represent genuinely independent realizations, based on different genetic precursors.

The adaptation of proteins for novel functions often requires changes in their kinetics via amino acid replacement. This process can require multiple mutations, and therefore extended periods of selection. The transfer of genes among distinct species might speed up the process, by providing proteins already adapted for the novel function. However, this hypothesis remains untested in multicellular eukaryotes. The grass Alloteropsis is an ideal system to test this hypothesis due to its diversity of genes encoding phosphoenolpyruvate carboxylase, an enzyme that catalyzes one of the key reactions in the C4 pathway. Different accessions of Alloteropsis either use native isoforms relatively recently co-opted from other functions or isoforms that were laterally acquired from distantly related species that evolved the C4 trait much earlier. By comparing the enzyme kinetics, we show that native isoforms with few amino acid replacements have substrate KM values similar to the non-C4 ancestral form, but exhibit marked increases in catalytic efficiency. The co-option of native isoforms was therefore followed by rapid catalytic improvements, which appear to rely on standing genetic variation observed within one species. Native C4 isoforms with more amino acid replacements exhibit additional changes in affinities, suggesting that the initial catalytic improvements are followed by gradual modifications. Finally, laterally acquired genes show both strong increases in catalytic efficiency and important changes in substrate handling. We conclude that the transfer of genes among distant species sharing the same physiological novelty creates an evolutionary shortcut toward more efficient enzymes, effectively accelerating evolution.