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Absorption of macronutrients such as nitrogen is a critical process for land plants. There is little information available on the correlation between the root evolution of land plants and the protein regulation of nitrogen absorption and responses. NIN-like protein (NLP) transcription factors contain a Phox and Bem1 (PB1) domain, which may regulate nitrate-response genes and seem to be involved in the adaptation to growing on land in terms of plant root development. In this report, we reveal the NLP phylogeny in land plants and the origin of NLP genes that may be involved in the nitrate-signaling pathway. Our NLP phylogeny showed that duplication of NLP genes occurred before divergence of chlorophyte and land plants. Duplicated NLP genes may lost in most chlorophyte lineages. The NLP genes of bryophytes were initially monophyletic, but this was followed by divergence of lycophyte NLP genes and then angiosperm NLP genes. Among those identified NLP genes, PB1, a protein–protein interaction domain was identified across our phylogeny. To understand how protein–protein interaction mediate via PB1 domain, we examined the PB1 domain of Arabidopsis thaliana NLP7 (AtNLP7) in terms of its molecular oligomerization and function as representative. Based on the structure of the PB1 domain, determined using small-angle x-ray scattering (SAXS) and site-directed mutagenesis, we found that the NLP7 PB1 protein forms oligomers and that several key residues (K867 and D909/D911/E913/D922 in the OPCA motif) play a pivotal role in the oligomerization of NLP7 proteins. The fact that these residues are all conserved across land plant lineages means that this oligomerization may have evolved after the common ancestor of extant land plants colonized the land. It would then have rapidly become established across land-plant lineages in order to mediate protein–protein interactions in the nitrate-signaling pathway.

Floral bilateral symmetry is one of the most important acquisitions in flower shape evolution in angiosperms. Members of Gesneriaceae possess predominantly zygomorphic flowers yet natural reversal to actinomorphy have independently evolved multiple times. The development of floral bilateral symmetry relies greatly on the gene CYCLOIDEA (CYC). Our reconstructed GCYC phylogeny indicated at least five GCYC duplication events occurred over the evolutionary history of Gesneriaceae. However, the patterns of GCYC expression following the duplications and the role of natural selection on GCYC copies in relation to floral symmetry remained largely unstudied. The Asiatic tribe Trichosporeae contains most reversals to actinomorphy. We thus investigated shifts in GCYC gene expression among selected zygomorphic species (Hemiboea bicornuta and Lysionotus pauciflorus) and species with reversals to actinomorphy (Conandron ramondioides) by RT-PCR. In the actinomorphic C. ramondioides, none of the three copies of GCYC was found expressed in petals implying that the reversal was a loss-of-function event. On the other hand, both zygomorphic species retained one GCYC1 copy that was expressed in the dorsal petals but each species utilized a different copy (GCYC1C for H. bicornuta and GCYC1D for L. pauciflorus). Together with previously published data, it appeared that GCYC1C and GCYC1D copies diversified their expression in a distinct species-specific pattern. To detect whether the selection signal (ω) changed before and after the duplication of GCYC1 in Asiatic Trichosporeae, we reconstructed a GCYC phylogeny using maximum likelihood and Bayesian inference algorithms and examined selection signals using PAML. The PAML analysis detected relaxation from selection right after the GCYC1 duplication (ωpre-duplication = 0.2819, ωpost-duplication = 0.3985) among Asiatic Trichosporeae species. We propose that the selection relaxation after the GCYC1 duplication created an "evolutionary window of flexibility" in which multiple copies were retained with randomly diverged roles for dorsal-specific expressions in association with floral symmetry changes.

Background Bilateral symmetry flower (zygomorphy) is the ancestral state for Gesneriaceae species. Yet independent reversions to actinomorphy have been parallelly evolved in several lineages. Conandron ramondioides is a natural radially symmetrical species survived in dense shade mountainous habitats where specialist pollinators are scarce. Whether the mutations in floral symmetry genes such as CYC, RAD and DIV genes, or their expression pattern shifts contribute to the reversion to actinomorphy in C. ramondioides thus facilitating shifts to generalist pollinators remain to be investigated. To address this, we isolated putative orthologues of these genes and relate their expressions to developmental stages of flower actinomorphy. Results Tissue specific RT-PCR found no dorsal identity genes CrCYCs and CrRADs expression in petal and stamen whorls, while the ventral identity gene CrDIV was expressed in all petals. Thus, ventralized actinomorphy is evolved in C. ramondioides. However, CrCYCs still persists their expression in sepal whorl. This is congruent with previous findings that CYC expression in sepals is an ancestral state common to both actinomorphic and zygomorphic core Eudicot species. Conclusions The loss of dorsal identity genes CrCYCs and CrRADs expression in petal and stamen whorl without mutating these genes specifies that a novel regulation change, possibly on cis-elements of these genes, has evolved to switch zygomorphy to actinomorphy. Electronic supplementary material The online version of this article (10.1186/s40529-018-0242-x) contains supplementary material, which is available to authorized users.