Comparative genomics has revealed that members of early divergent lineages of land plants share a set of highly conserved transcription factors (TFs) with flowering plants. While gene copy numbers have expanded through time, it has been predicted that diversification, co-option, and reassembly of gene regulatory networks implicated in development are directly related to morphological innovations that led to more complex land plant bodies. Examples of key networks have been deeply studied in Arabidopsis thaliana, such as those involving the AINTEGUMENTA (ANT) gene family that encodes AP2-type TFs. These TFs play significant roles in plant development such as the maintenance of stem cell niches, the correct development of the embryo and the formation of lateral organs, as well as fatty acid metabolism. Previously, it has been hypothesized that the common ancestor of mosses and vascular plants encoded two ANT genes that later diversified in seed plants. However, algae and bryophyte sequences have been underrepresented from such phylogenetic analyses. To understand the evolution of ANT in a complete manner, we performed phylogenetic analyses of ANT protein sequences of representative species from across the Streptophyta clade, including algae, liverworts, and hornworts, previously unrepresented. Moreover, protein domain architecture, selection analyses, and regulatory cis elements prediction, allowed us to propose a scenario of how the evolution of ANT genes occurred. In this study we show that a duplication of a preANT-like gene in the ancestor of embryophytes may have given rise to the land plant-exclusive basalANT and euANT lineages. We hypothesize that the absence of euANT-type and basalANT-type sequences in algae, and its presence in extant land plant species, suggests that the divergence of pre-ANT into basal and eu-ANT clades in embryophytes may have influenced the conquest of land by plants, as ANT TFs play important roles in tolerance to desiccation and the establishment, maintenance, and development of complex multicellular structures which either became more complex or appeared in land plants.

Phosphate (Pi) is a pivotal nutrient that constraints plant development and productivity in natural ecosystems. Land colonization by plants, more than 470 million years ago, evolved adaptive mechanisms to conquer Pi-scarce environments. However, little is known about the molecular basis underlying such adaptations at early branches of plant phylogeny. To shed light on how early divergent plants respond to Pi limitation, we analyzed the morpho-physiological and transcriptional dynamics of Marchantia polymorpha upon Pi starvation. Our phylogenomic analysis highlights some gene networks present since the Chlorophytes and others established in the Streptophytes (e.g., PHR1–SPX1 and STOP1–ALMT1, respectively). At the morpho-physiological level, the response is characterized by the induction of phosphatase activity, media acidification, accumulation of auronidins, reduction of internal Pi concentration, and developmental modifications of rhizoids. The transcriptional response involves the induction of MpPHR1, Pi transporters, lipid turnover enzymes, and MpMYB14, which is an essential transcription factor for auronidins biosynthesis. MpSTOP2 up-regulation correlates with expression changes in genes related to organic acid biosynthesis and transport, suggesting a preference for citrate exudation. An analysis of MpPHR1 binding sequences (P1BS) shows an enrichment of this cis regulatory element in differentially expressed genes. Our study unravels the strategies, at diverse levels of organization, exerted by M. polymorpha to cope with low Pi availability.

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Phosphate (Pi) is a pivotal nutrient that constraints plant development and productivity in natural ecosystems. Land colonization by plants, more than 470 million years ago, evolved adaptive mechanisms to conquer Pi-scarce environments. However, little is known about the molecular basis underlying such adaptations at early branches of plant phylogeny. To shed light on how early divergent plants respond to Pi limitation, we analyzed the morpho-physiological and transcriptional dynamics of Marchantia polymorpha upon Pi starvation. Our phylogenomic analysis highlights some gene networks present since the Chlorophytes and others established in the Streptophytes (eg. PHR1-SPX1 and STOP1-ALMT1, respectively). At the morpho-physiological level, the response is characterized by the induction of phosphatase activity, media acidification, accumulation of auronidins, reduction of internal Pi concentration and developmental modifications of rhizoids. The transcriptional response involves the induction of MpPHR1, Pi transporters, lipid turnover enzymes and MpMYB14, an essential transcription factor for auronidins biosynthesis. MpSTOP2 up-regulation correlates with expression changes in genes related to organic acid biosynthesis and transport, suggesting preference for citrate exudation. Analysis of MpPHR1 binding sequences (P1BS) shows enrichment of this cis regulatory element in differentially expressed genes. Our study unravels the strategies, at diverse levels of organization, exerted by M. polymorpha to cope with low Pi availability.Significance StatementThis study unravels the transcriptional and morphophysiological mechanisms executed by the non-vascular, and rootless, plant Marchantia polymorpha upon phosphate starvation conditions. The findings in this study shed light on the mechanisms that early land plants may have developed for the conquest of substrates poor in available phosphate, some of which are still conserved by current-day plants. Moreover, our results open several working hypotheses and novel perspectives for the study of Pi-starvation responses along plant evolution.

Plant hormones are master regulators of developmental and genetic mechanisms to deal with diverse environmental cues. Upon phosphate (Pi) limitation, vascular plants modify phytohormone metabolism to coordinate diverse mechanisms to overcome such stress. However, the transcriptional program underlying hormonal signaling in response to Pi scarcity in early branches of land plant phylogeny, remains unclear. Therefore, we explored the transcriptional dynamics of key genes involved in auxin, cytokinin, ethylene, jasmonate, gibberellin and abscisic acid metabolism in the early divergent land plant Marchantia polymorpha, upon Pi starvation. Our RNAseq approach revealed major changes in genes associated with auxin and ethylene biosynthesis. Genes involved in cytokinin synthesis are repressed. Interestingly, genes involved in auxin and ethylene signaling such as MpARF1 and MpARF2 are upregulated. In contrast, MpARRb is down-regulated. Moreover, genes involved in the synthesis of jasmonates were highly upregulated, but those related to signaling did not change in expression. Our data suggest that auxin and ethylene act as positive regulators of rhizoid development under Pi-limited conditions, whereas cytokinin may act as a negative regulator. The transcriptional behaviour of some hormonerelated genes in Marchantia is similar to those described in controlling root hair development in Arabidopsis, Maize and Rice, upon Pi scarcity.