Structural insights into AtABCG25, an angiosperm-specific abscisic acid exporter.

Cellular hormone homeostasis is essential for precise spatial and temporal signaling responses and plant fitness. Abscisic acid (ABA) plays pivotal roles in orchestrating various developmental and stress responses and confers fitness benefits over ecological and evolutionary timescales in terrestrial plants. Cellular ABA level is regulated by complex processes, including biosynthesis, catabolism, and transport. AtABCG25 is the first ABA exporter identified through genetic screening and affects diverse ABA responses. Resolving the structural basis of ABA export by ABCG25 is critical for further manipulations of ABA homeostasis and plant fitness. We used cryo-electron microscopy to elucidate the structural dynamics of AtABCG25 and successfully characterized different states, including apo AtABCG25, ABA-bound AtABCG25, and ATP-bound AtABCG25 (E232Q). Notably, AtABCG25 forms a homodimer that features a deep, slit-like cavity in the transmembrane domain, and we precisely characterized the critical residues in the cavity where ABA binds. ATP binding triggers closure of the nucleotide-binding domains and conformational transitions in the transmembrane domains. We show that AtABCG25 belongs to a conserved ABCG subfamily that originated during the evolution of angiosperms. This subfamily neofunctionalized to regulate seed germination via the endosperm, in concert with the evolution of this angiosperm-specific, embryo-nourishing tissue. Collectively, these findings provide valuable insights into the intricate substrate recognition and transport mechanisms of the ABA exporter AtABCG25, paving the way for genetic manipulation of ABA homeostasis and plant fitness.

Updated Phylogeny and Protein Structure Predictions Revise the Hypothesis on the Origin of MADS-box Transcription Factors in Land Plants.

MADS-box transcription factors (TFs), among the first TFs extensively studied, exhibit a wide distribution across eukaryotes and play diverse functional roles. Varying by domain architecture, MADS-box TFs in land plants are categorized into Type I (M-type) and Type II (MIKC-type). Type I and II genes have been considered orthologous to the SRF and MEF2 genes in animals, respectively, presumably originating from a duplication before the divergence of eukaryotes. Here, we exploited the increasing availability of eukaryotic MADS-box sequences and reassessed their evolution. While supporting the ancient duplication giving rise to SRF- and MEF2-types, we found that Type I and II genes originated from the MEF2-type genes through another duplication in the most recent common ancestor (MRCA) of land plants. Protein structures predicted by AlphaFold2 and OmegaFold support our phylogenetic analyses, with plant Type I and II TFs resembling the MEF2-type structure, rather than SRFs. We hypothesize that the ancestral SRF-type TFs were lost in the MRCA of Archaeplastida (the kingdom Plantae sensu lato). The retained MEF2-type TFs acquired a Keratin-like domain and became MIKC-type before the divergence of Streptophyta. Subsequently in the MRCA of land plants, M-type TFs evolved from a duplicated MIKC-type precursor through loss of the Keratin-like domain, leading to the Type I clade. Both Type I and II TFs expanded and functionally differentiated in concert with the increasing complexity of land plant body architecture. The recruitment of these originally stress-responsive TFs into developmental programs, including those underlying reproduction, may have facilitated the adaptation to the terrestrial environment.

The Saururus chinensis genome provides insights into the evolution of pollination strategies and herbaceousness in magnoliids.

Saururus chinensis, an herbaceous magnoliid without perianth, represents a clade of early-diverging angiosperms that have gone through woodiness-herbaceousness transition and pollination obstacles: the characteristic white leaves underneath inflorescence during flowering time are considered a substitute for perianth to attract insect pollinators. Here, using the newly sequenced S. chinensis genome, we revisited the phylogenetic position of magnoliids within mesangiosperms, and recovered a sister relationship for magnoliids and Chloranthales. By considering differentially expressed genes, we identified candidate genes that are involved in the morphogenesis of the white leaves in S. chinensis. Among those genes, we verified - in a transgenic experiment with Arabidopsis - that increasing the expression of the "pseudo-etiolation in light" gene (ScPEL) can inhibit the biosynthesis of chlorophyll. ScPEL is thus likely responsible for the switches between green and white leaves, suggesting that changes in gene expression may underlie the evolution of pollination strategies. Despite being an herbaceous plant, S. chinensis still has vascular cambium and maintains the potential for secondary growth as a woody plant, because the necessary machinery, i.e., the entire gene set involved in lignin biosynthesis, is well preserved. However, similar expression levels of two key genes (CCR and CAD) between the stem and other tissues in the lignin biosynthesis pathway are possibly associated with the herbaceous nature of S. chinensis. In conclusion, the S. chinensis genome provides valuable insights into the adaptive evolution of pollination in Saururaceae and reveals a possible mechanism for the evolution of herbaceousness in magnoliids.

Endosperm Evolution by Duplicated and Neofunctionalized Type I MADS-Box Transcription Factors.

MADS-box transcription factors (TFs) are present in nearly all major eukaryotic groups. They are divided into Type I and Type II that differ in domain structure, functional roles, and rates of evolution. In flowering plants, major evolutionary innovations like flowers, ovules, and fruits have been closely connected to Type II MADS-box TFs. The role of Type I MADS-box TFs in angiosperm evolution remains to be identified. Here, we show that the formation of angiosperm-specific Type I MADS-box clades of Mγ and Mγ-interacting Mα genes (Mα*) can be tracked back to the ancestor of all angiosperms. Angiosperm-specific Mγ and Mα* genes were preferentially expressed in the endosperm, consistent with their proposed function as heterodimers in the angiosperm-specific embryo nourishing endosperm tissue. We propose that duplication and diversification of Type I MADS genes underpin the evolution of the endosperm, a developmental innovation closely connected to the origin and success of angiosperms.

H2A ubiquitination is essential for Polycomb Repressive Complex 1-mediated gene regulation in Marchantia polymorpha.

BACKGROUND: Polycomb repressive complex 1 (PRC1) and PRC2 are chromatin regulators maintaining transcriptional repression. The deposition of H3 lysine 27 tri-methylation (H3K27me3) by PRC2 is known to be required for transcriptional repression, whereas the contribution of H2A ubiquitination (H2Aub) in the Polycomb repressive system remains unclear in plants. RESULTS: We directly test the requirement of H2Aub for gene regulation in Marchantia polymorpha by generating point mutations in H2A that prevent ubiquitination by PRC1. These mutants show reduced H3K27me3 levels on the same target sites as mutants defective in PRC1 subunits MpBMI1 and the homolog MpBMI1L, revealing that PRC1-catalyzed H2Aub is essential for Polycomb system function. Furthermore, by comparing transcriptome data between mutants in MpH2A and MpBMI1/1L, we demonstrate that H2Aub contributes to the PRC1-mediated transcriptional level of genes and transposable elements. CONCLUSION: Together, our data demonstrates that H2Aub plays a direct role in H3K27me3 deposition and is required for PRC1-mediated transcriptional changes in both genes and transposable elements in Marchantia.

Mobility connects: transposable elements wire new transcriptional networks by transferring transcription factor binding motifs.

Transposable elements (TEs) constitute major fractions of plant genomes. Their potential to be mobile provides them with the capacity to cause major genome rearrangements. Those effects are potentially deleterious and enforced the evolution of epigenetic suppressive mechanisms controlling TE activity. However, beyond their deleterious effects, TE insertions can be neutral or even advantageous for the host, leading to long-term retention of TEs in the host genome. Indeed, TEs are increasingly recognized as major drivers of evolutionary novelties by regulating the expression of nearby genes. TEs frequently contain binding motifs for transcription factors and capture binding motifs during transposition, which they spread through the genome by transposition. Thus, TEs drive the evolution and diversification of gene regulatory networks by recruiting lineage-specific targets under the regulatory control of specific transcription factors. This process can explain the rapid and repeated evolution of developmental novelties, such as C4 photosynthesis and a wide spectrum of stress responses in plants. It also underpins the convergent evolution of embryo nourishing tissues, the placenta in mammals and the endosperm in flowering plants. Furthermore, the gene regulatory network underlying flower development has also been largely reshaped by TE-mediated recruitment of regulatory elements; some of them being preserved across long evolutionary timescales. In this review, we highlight the potential role of TEs as evolutionary toolkits in plants by showcasing examples of TE-mediated evolutionary novelties.

MEKK2 inhibits activation of MAP kinases in Arabidopsis.

The Arabidopsis MEKK1-MKK1/MKK2-MPK4 kinase cascade is monitored by the nucleotide-binding leucine-rich-repeat immune receptor SUMM2. Disruption of this kinase cascade leads to activation of SUMM2-mediated immune responses. MEKK2, a close paralog of MEKK1, is required for defense responses mediated by SUMM2, the molecular mechanism of which is unclear. In this study, we showed that MEKK2 serves as a negative regulator of MPK4. It binds to MPK4 to directly inhibit its phosphorylation by upstream MKKs. Activation of SUMM2-mediated defense responses induces the expression of MEKK2, which in turn blocks MPK4 phosphorylation to further amplify immune responses mediated by SUMM2. Intriguingly, MEKK2 locates in a tandem repeat consisting of MEKK1, MEKK2 and MEKK3, which was generated from a recent gene duplication event, suggesting that MEKK2 evolved from a MAPKKK to become a negative regulator of MAP kinases.

Divergence of duplicated genes by repeated partitioning of splice forms and subcellular localization.

Gene duplication is a prominent and recurrent process in plant genomes. Among the possible fates of duplicated genes, subfunctionalization refers to duplicates taking on different parts of the function or expression pattern of the ancestral gene. This partitioning could be accompanied by changes in subcellular localization of the protein products. When alternative splicing of gene products leads to protein products with different subcellular localizations, we propose that after gene duplication there will be partitioning of the alternatively spliced forms such that the products of each duplicate are localized to only one of the original locations, which we refer to as sublocalization. We identified the plastid ascorbate peroxidase (cpAPX) genes across angiosperms and analyzed their duplication history, alternative splicing, and subcellular targeting patterns to identify cases of sublocalization. We found angiosperms typically have one cpAPX gene that generates both thylakoidal APX (tAPX) and stromal APX (sAPX) through alternative splicing. We identified several independent lineage-specific sublocalization cases with specialized paralogues of tAPX and sAPX. We determined that the sublocalization happened through two types of sequence evolution patterns. Our findings suggest that the divergence through sublocalization is key to the retention of paralogous cpAPX genes in angiosperms.

The MADS-box transcription factor PHERES1 controls imprinting in the endosperm by binding to domesticated transposons.

MADS-box transcription factors (TFs) are ubiquitous in eukaryotic organisms and play major roles during plant development. Nevertheless, their function in seed development remains largely unknown. Here, we show that the imprinted Arabidopsis thaliana MADS-box TF PHERES1 (PHE1) is a master regulator of paternally expressed imprinted genes, as well as of non-imprinted key regulators of endosperm development. PHE1 binding sites show distinct epigenetic modifications on maternal and paternal alleles, correlating with parental-specific transcriptional activity. Importantly, we show that the CArG-box-like DNA-binding motifs that are bound by PHE1 have been distributed by RC/Helitron transposable elements. Our data provide an example of the molecular domestication of these elements which, by distributing PHE1 binding sites throughout the genome, have facilitated the recruitment of crucial endosperm regulators into a single transcriptional network.

Concerted Divergence after Gene Duplication in Polycomb Repressive Complexes.

Duplicated genes are a major contributor to genome evolution and phenotypic novelty. There are multiple possible evolutionary fates of duplicated genes. Here, we provide an example of concerted divergence of simultaneously duplicated genes whose products function in the same complex. We studied POLYCOMB REPRESSIVE COMPLEX2 (PRC2) in Brassicaceae. The VERNALIZATION (VRN)-PRC2 complex contains VRN2 and SWINGER (SWN), and both genes were duplicated during a whole-genome duplication to generate FERTILIZATION INDEPENDENT SEED2 (FIS2) and MEDEA (MEA), which function in the Brassicaceae-specific FIS-PRC2 complex that regulates seed development. We examined the expression of FIS2, MEA, and their paralogs, compared their cytosine and histone methylation patterns, and analyzed the sequence evolution of the genes. We found that FIS2 and MEA have reproductive-specific expression patterns that are correlated and derived from the broadly expressed VRN2 and SWN in outgroup species. In vegetative tissues of Arabidopsis (Arabidopsis thaliana), repressive methylation marks are enriched in FIS2 and MEA, whereas active marks are associated with their paralogs. We detected comparable accelerated amino acid substitution rates in FIS2 and MEA but not in their paralogs. We also show divergence patterns of the PRC2-associated VERNALIZATION5/VIN3-LIKE2 that are similar to FIS2 and MEA These lines of evidence indicate that FIS2 and MEA have diverged in concert, resulting in functional divergence of the PRC2 complexes in Brassicaceae. This type of concerted divergence is a previously unreported fate of duplicated genes. In addition, the Brassicaceae-specific FIS-PRC2 complex modified the regulatory pathways in female gametophyte and seed development.

Frequent changes in expression profile and accelerated sequence evolution of duplicated imprinted genes in arabidopsis.

Eukaryotic genomes have large numbers of duplicated genes that can evolve new functions or expression patterns by changes in coding and regulatory sequences, referred to as neofunctionalization. In flowering plants, some duplicated genes are imprinted in the endosperm, where only one allele is expressed depending on its parental origin. We found that 125 imprinted genes in Arabidopsis arose from gene duplication events during the evolution of the Brassicales. Analyses of 46 gene pairs duplicated by an ancient whole-genome duplication (alpha WGD) indicated that many imprinted genes show an accelerated rate of amino acid changes compared with their paralogs. Analyses of microarray expression data from 63 organ types and developmental stages indicated that many imprinted genes have expression patterns restricted to flowers and/or seeds in contrast to their broadly expressed paralogs. Assays of expression in orthologs from outgroup species revealed that some imprinted genes have acquired an organ-specific expression pattern restricted to flowers and/or seeds. The changes in expression pattern and the accelerated sequence evolution in the imprinted genes suggest that some of them may have undergone neofunctionalization. The imprinted genes MPC, HOMEODOMAIN GLABROUS6 (HDG6), and HDG3 are particularly interesting cases that have different functions from their paralogs. This study indicates that a large number of imprinted genes in Arabidopsis are evolutionarily recent duplicates and that many of them show changes in expression profiles and accelerated sequence evolution. Acquisition of imprinting is a mode of duplicate gene divergence in plants that is more common than previously thought.