Title: Bona Fide Plant Steroid Receptors Are Innovated in Seed Plants and Angiosperms through Successive Whole Genome Duplication Events.

Whole genome duplication (WGD) events are widespread in plants and animals, thus their long-term evolutionary contribution has long been speculated, yet a specific contribution is difficult to verify. Here, we show that ɛ-WGD and ζ-WGD contribute to the origin and evolution of bona fide brassinosteroid (BR) signaling through the innovation of active BR biosynthetic enzymes and active BR receptors from their respective ancestors. We found that BR receptors BRI1 (BR Insensitive 1) and BRL1/3 (BRI1-likes 1/3) derived by ɛ-WGD and ζ-WGD, which occurred in the common ancestor of angiosperms and seed plants, respectively, while orphan BR receptor BRL2 first appeared in stomatophytes. Additionally, CYP85A enzymes synthesizing the bioactive BRs derived from a common ancestor of seed plants while its sister enzymes CYP90 synthesizing BR precursors presented in all land plants, implying possible ligand-receptor coevolution. Consistently, the island domains (IDs) responsible for BR perception in BR receptors were most divergent among different receptor branches, supporting ligand-driven evolution. As a result, BRI1 was the most diversified BR receptor in angiosperms. Importantly, relative to the BR biosynthetic DET2 gene presented in all land plants, BRL2, BRL1/3 and BRI1 had high expression in vascular plants ferns, gymnosperms and angiosperms, respectively. Notably, BRI1 is the most diversified BR receptor with the most abundant expression in angiosperms, suggesting potential positive selection. Therefore, WGDs initiate a neofunctionalization process diverged by ligand-perception and transcriptional expression, which might optimize both BR biosynthetic enzymes and BR receptors, likely contributing to the evolution of land plants, especially seed plants and angiosperms.

Kinase regulators evolved into two families by gain and loss of ability to bind plant steroid receptors.

All biological functions evolve by fixing beneficial mutations and removing deleterious ones. Therefore, continuously fixing and removing the same essential function to separately diverge monophyletic gene families sounds improbable. Yet, here we report that brassinosteroid insensitive1 kinase inhibitor1 (BKI1)/membrane-associated kinase regulators (MAKRs) regulating a diverse function evolved into BKI1 and MAKR families from a common ancestor by respectively enhancing and losing ability to bind brassinosteroid receptor brassinosteroid insensitive1 (BRI1). The BKI1 family includes BKI1, MAKR1/BKI1-like (BKL) 1, and BKL2, while the MAKR family contains MAKR2-6. Seedless plants contain only BKL2. In seed plants, MAKR1/BKL1 and MAKR3, duplicates of BKL2, gained and lost the ability to bind BRI1, respectively. In angiosperms, BKL2 lost the ability to bind BRI1 to generate MAKR2, while BKI1 and MAKR6 were duplicates of MAKR1/BKL1 and MAKR3, respectively. In dicots, MAKR4 and MAKR5 were duplicates of MAKR3 and MAKR2, respectively. Importantly, BKI1 localized in the plasma membrane, but BKL2 localized to the nuclei while MAKR1/BKL1 localized throughout the whole cell. Importantly, BKI1 strongly and MAKR1/BKL1 weakly inhibited plant growth, but BKL2 and the MAKR family did not inhibit plant growth. Functional study of the chimeras of their N- and C-termini showed that only the BKI1 family was partially reconstructable, supporting stepwise evolution by a seesaw mechanism between their C- and N-termini to alternately gain an ability to bind and inhibit BRI1, respectively. Nevertheless, the C-terminal BRI1-interacting motif best defines the divergence of BKI1/MAKRs. Therefore, BKI1 and MAKR families evolved by gradually gaining and losing the same function, respectively, extremizing divergent evolution and adding insights into gene (BKI1/MAKR) duplication and divergence.