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The appearance of plant organs mediated the explosive radiation of land plants, which shaped the biosphere and allowed the establishment of terrestrial animal life. The evolution of organs and immobile gametes required the coordinated acquisition of novel gene functions, the co-option of existing genes and the development of novel regulatory programmes. However, no large-scale analyses of genomic and transcriptomic data have been performed for land plants. To remedy this, we generated gene expression atlases for various organs and gametes of ten plant species comprising bryophytes, vascular plants, gymnosperms and flowering plants. A comparative analysis of the atlases identified hundreds of organ- and gamete-specific orthogroups and revealed that most of the specific transcriptomes are significantly conserved. Interestingly, our results suggest that co-option of existing genes is the main mechanism for evolving new organs. In contrast to female gametes, male gametes showed a high number and conservation of specific genes, which indicates that male reproduction is highly specialized. The expression atlas capturing pollen development revealed numerous transcription factors and kinases essential for pollen biogenesis and function. Ministry of Education (MOE) Nanyang Technological University Submitted/Accepted version I.J. is supported by Singaporean Ministry of Education grant MOE2018-T2-2-053, while M.M. is supported by a NTU Start-Up Grant. ERA-CAPS EVO-REPRO I2163 and FWF grant P30802 were awarded to F.B.; FCT ERA-CAPS-0001-2014 and PTDC-BIA-FBT-28484-2017 to J.D.B.; and ERA-CAPS EVO-REPRO DR 334/12-1 to S.S. and T.D. D. Hackenberg was supported by ERA-CAPS UK Biotechnology and Biological Research Council grant BB/N005090 awarded to D.T.; M.B. was supported through the FWF Lise Meitner fellowship M1818. The Vienna BioCenter Core Facilities GmbH (VBCF) Plant Sciences Facility acknowledges funding from the Austrian Federal Ministry of Education, Science and Research and the City of Vienna. L.S. was supported by CSF grant 17-23183S. C.M. and D. Honys were supported by the Czech Ministry of Education, Youth and Sport (LTC18034 and LTAIN19030) through the European Regional Development Fund-Project “Centre for Experimental Plant Biology” number CZ.02.1.01/0.0/0.0/16_019/0000738. The Genomics Unit of Instituto Gulbenkian de Ciência was partially supported by the ONEIDA Project (LISBOA01-0145-FEDER-016417) co-funded by FEEI–‘Fundos Europeus Estruturais e de Investimento’ from the ‘Programa Operacional Regional Lisboa 2020’ and by national funds from FCT–‘Fundação para a Ciência e a Tecnologia’. C.S.M. acknowledges a doctoral fellowship from the FCT (PD/BD/114362/2016) under the Plants for Life PhD Program. J.D.B. received salary support from the FCT through an ‘Investigador FCT’ position. M.J. and J.G. were supported by a US National Science Foundation grant (IOS1540019).

The evolution of plant organs, including leaves, stems, roots, and flowers, mediated the explosive radiation of land plants, which shaped the biosphere and allowed the establishment of terrestrial animal life. Furthermore, the fertilization products of angiosperms, seeds serve as the basis for most of our food. The evolution of organs and immobile gametes required the coordinated acquisition of novel gene functions, the co-option of existing genes, and the development of novel regulatory programs. However, our knowledge of these events is limited, as no large-scale analyses of genomic and transcriptomic data have been performed for land plants. To remedy this, we have generated gene expression atlases for various organs and gametes of 10 plant species comprising bryophytes, vascular plants, gymnosperms, and flowering plants. Comparative analysis of the atlases identified hundreds of organ- and gamete-specific gene families and revealed that most of the specific transcriptomes are significantly conserved. Interestingly, the appearance of organ-specific gene families does not coincide with the corresponding organ’s appearance, suggesting that co-option of existing genes is the main mechanism for evolving new organs. In contrast to female gametes, male gametes showed a high number and conservation of specific genes, suggesting that male reproduction is highly specialized. The expression atlas capturing pollen development revealed numerous transcription factors and kinases essential for pollen biogenesis and function. To provide easy access to the expression atlases and these comparative analyses, we provide an online database, www.evorepro.plant.tools, that allows the exploration of expression profiles, organ-specific genes, phylogenetic trees, co-expression networks, and others.

Key messagePartially dominant resistance to Turnip yellows virus associated with one major QTL was identified in the natural allotetraploid oilseed rape cultivar Yudal.AbstractTurnip yellows virus (TuYV) is transmitted by the peach-potato aphid (Myzus persicae) and causes severe yield losses in commercial oilseed rape crops (Brassica napus). There is currently only one genetic resource for resistance to TuYV available in brassica, which was identified in the re-synthesisedB. napusline ‘R54’. In our study, 27 mostly homozygousB. napusaccessions, either doubled-haploid (DH) or inbred lines, representing a diverse subset of theB. napusgenepool, were screened for TuYV resistance/susceptibility. Partial resistance to TuYV was identified in the Korean spring oilseed rape,B. napusvariety Yudal, whilst the dwarf French winter oilseed rape line Darmor-bzhwas susceptible. QTL mapping using the established Darmor-bzh× Yudal DH mapping population (DYDH) revealed one major QTL explaining 36% and 18% of the phenotypic variation in two independent experiments. A DYDH line was crossed to Yudal, and reciprocal backcross (BC1) populations from the F1with either the susceptible or resistant parent revealed the dominant inheritance of the TuYV resistance. The QTL on ChrA04 was verified in the segregating BC1population. A second minor QTL on ChrC05 was identified in one of the two DYDH experiments, and it was not observed in the BC1population. The TuYV resistance QTL in ‘R54’ is within the QTL interval on Chr A04 of Yudal; however, the markers co-segregating with the ‘R54’ resistance are not conserved in Yudal, suggesting an independent origin of the TuYV resistances. This is the first report of the QTL mapping of TuYV resistance in naturalB. napus.

The reproductive adaptations of land plants have played a key role in their terrestrial colonization and radiation. This encompasses mechanisms used for the production, dispersal and union of gametes to support sexual reproduction. The production of small motile male gametes and larger immotile female gametes (oogamy) in specialized multicellular gametangia evolved in the charophyte algae, the closest extant relatives of land plants. Reliance on water and motile male gametes for sexual reproduction was retained by bryophytes and basal vascular plants, but was overcome in seed plants by the dispersal of pollen and the guided delivery of non-motile sperm to the female gametes. Here we discuss the evolutionary history of male gametogenesis in streptophytes (green plants) and the underlying developmental biology, including recent advances in bryophyte and angiosperm models. We conclude with a perspective on research trends that promise to deliver a deeper understanding of the evolutionary and developmental mechanisms of male gametogenesis in plants.

Signaling pathways regulated by heterotrimeric G-proteins exist in all eukaryotes. The regulator of G-protein signaling (RGS) proteins are key interactors and critical modulators of the Gα protein of the heterotrimer. However, while G-proteins are widespread in plants, RGS proteins have been reported to be missing from the entire monocot lineage, with two exceptions. A single amino acid substitution-based adaptive coevolution of the Gα:RGS proteins was proposed to enable the loss of RGS in monocots. We used a combination of evolutionary and biochemical analyses and homology modeling of the Gα and RGS proteins to address their expansion and its potential effects on the G-protein cycle in plants. Our results show that RGS proteins are widely distributed in the monocot lineage, despite their frequent loss. There is no support for the adaptive coevolution of the Gα:RGS protein pair based on single amino acid substitutions. RGS proteins interact with, and affect the activity of, Gα proteins from species with or without endogenous RGS. This cross-functional compatibility expands between the metazoan and plant kingdoms, illustrating striking conservation of their interaction interface. We propose that additional proteins or alternative mechanisms may exist which compensate for the loss of RGS in certain plant species.

The eukaryotic transcription factor NF-Y consists of three subunits (A, B, and C), which are encoded in Arabidopsis thaliana in multigene families consisting of 10, 13, and 13 genes, respectively. In principle, all potential combinations of the subunits are possible for the assembly of the heterotrimeric complex. We aimed at assessing the probability of each subunit to participate in the assembly of NF-Y. The evaluation of physical interactions among all members of the NF-Y subunit families indicate a strong requirement for NF-YB/NF-YC heterodimerization before the entire complex can be accomplished. By means of a modified yeast two-hybrid system assembly of all three subunits to a heterotrimeric complex was demonstrated. Using GFP fusion constructs, NF-YA and NF-YC localization in the nucleus was demonstrated, while NF-YB is solely imported into the nucleus as a NF-YC-associated heterodimer NF-YC. This piggyback transport of the two Arabidopsis subunits differs from the import of the NF-Y heterotrimer of heterotrophic organisms. Based on a peptide structure model of the histone-fold-motifs, disulfide bonding among intramolecular conserved cysteine residues of NF-YB, which is responsible for the redox-regulated assembly of NF-YB and NF-YC in human and Aspergillus nidulans, can be excluded for Arabidopsis NF-YB.

The transcription factor NF-Y consists of the three subunits A, B and C, which are encoded in Arabidopsis in large gene families. The multiplicity of the genes implies that NF-Y may act in diverse combinations of each subunit for the transcriptional control. We aimed to assign a function in stress response and plant development to NF-YC subunits by analyzing the expression of NF-Y genes and exploitation of nf-y mutants. Among the subunit family, NF-YC2 showed the strongest inducibility towards oxidative stress, e.g. photodynamic, light, oxidative, heat and drought stress. A tobacco NF-YC homologous gene was found to be inducible by photooxidative stress generated by an accumulation of the tetrapyrrole metabolite, coproporphyrin. Despite the stress induction, an Arabidopsis nf-yc2 mutant and NF-YC2 overexpressors did not show phenotypical differences compared to wild-type seedlings in response to photooxidative stress. This can be explained by the compensatory potential of other members of the NF-YC family. However, NF-YC2 overexpression leads to an early flowering phenotype that is correlated with increased FLOWERING LOCUS T-transcript levels. It is proposed that NF-YC2 functions in floral induction and is a candidate gene among the NF-Y family for the transcriptional activation upon oxidative stress.

Evolutionary mechanisms underlying innovation of cell types have remained largely unclear. In multicellular eukaryotes, the evolutionary molecular origin of sperm differentiation is unknown in most lineages. Here, we report that in algal ancestors of land plants, changes in the DNA-binding domain of the ancestor of the MYB transcription factor DUO1 enabled the recognition of a new cis-regulatory element. This event led to the differentiation of motile sperm. After neo-functionalization, DUO1 acquired sperm lineage-specific expression in the common ancestor of land plants. Subsequently the downstream network of DUO1 was rewired leading to sperm with distinct morphologies. Conjugating green algae, a sister group of land plants, accumulated mutations in the DNA-binding domain of DUO1 and lost sperm differentiation. Our findings suggest that the emergence of DUO1 was the defining event in the evolution of sperm differentiation and the varied modes of sexual reproduction in the land plant lineage., 植物の多様な精子の形成の進化的起源を解明 --7億年前のDUO1遺伝子獲得が植物の精子形成に関わる--. 京都大学プレスリリース. 2018-12-13.

The lack of heterotrimeric G-protein homologs in the sequenced genomes of green algae has led to the hypothesis that, in plants, this signaling mechanism coevolved with the embryophytic life cycle and the acquisition of terrestrial habitat. Given the large evolutionary gap that exists between the chlorophyte green algae and most basal land plants, the bryophytes, we evaluated the presence of this signaling complex in a charophyte green alga, Chara braunii, proposed to be the closest living relative of land plants. The C. braunii genome encodes for the entire G-protein complex, the Gα, Gβ, and Gγ subunits, and the REGULATOR OF G-PROTEIN SIGNALING (RGS) protein. The biochemical properties of these proteins and their cross-species functionality show that they are functional homologs of canonical G-proteins. The subunit-specific interactions between CbGα and CbGβ, CbGβ and CbGγ, and CbGα and CbRGS are also conserved, establishing the existence of functional G-protein complex-based signaling mechanisms in green algae.

Heterotrimeric G-proteins are important signaling intermediates in all eukaryotes. These proteins link signal perception by a cell surface localized receptor to the downstream effectors of a given signaling pathways. The minimal core of the heterotrimeric G-protein complex consists of Gα, Gβ, and Gγ subunits, the G protein coupled receptor (GPCR) and the regulator of G-protein signaling (RGS) proteins. Signal transduction by heterotrimeric G-proteins is controlled by the distinct biochemical activities of Gα protein, which binds and hydrolyses GTP. Evaluation of the rate of GTP binding, the rate of GTP hydrolysis, and the rate of GTP/GDP exchange on Gα protein are required to better understand the mechanistic aspects of heterotrimeric G-protein signaling, which remains significantly limited for the plant G-proteins. Here we describe the optimized methods for measurement of the distinct biochemical activities of the Arabidopsis Gα protein.

Heterotrimeric G-proteins are important signaling intermediates in all eukaryotes. These proteins link signal perception by a cell surface localized receptor to the downstream effectors of a given signaling pathways. The minimal core of the heterotrimeric G-protein complex consists of Gα, Gβ, and Gγ subunits, the G protein coupled receptor (GPCR) and the regulator of G-protein signaling (RGS) proteins. Signal transduction by heterotrimeric G-proteins is controlled by the distinct biochemical activities of Gα protein, which binds and hydrolyses GTP. Evaluation of the rate of GTP binding, the rate of GTP hydrolysis, and the rate of GTP/GDP exchange on Gα protein are required to better understand the mechanistic aspects of heterotrimeric G-protein signaling, which remains significantly limited for the plant G-proteins. Here we describe the optimized methods for measurement of the distinct biochemical activities of the Arabidopsis Gα protein.

In this study, we report the functional characterization of heterotrimeric G-proteins from a nonvascular plant, the moss Physcomitrella patens. In plants, G-proteins have been characterized from only a few angiosperms to date, where their involvement has been shown during regulation of multiple signaling and developmental pathways affecting overall plant fitness. In addition to its unparalleled evolutionary position in the plant lineages, the P. patens genome also codes for a unique assortment of G-protein components, which includes two copies of Gβ and Gγ genes, but no canonical Gα. Instead, a single gene encoding an extra-large Gα (XLG) protein exists in the P. patens genome. Here, we demonstrate that in P. patens the canonical Gα is biochemically and functionally replaced by an XLG protein, which works in the same genetic pathway as one of the Gβ proteins to control its development. Furthermore, the specific G-protein subunits in P. patens are essential for its life cycle completion. Deletion of the genomic locus of PpXLG or PpGβ2 results in smaller, slower growing gametophores. Normal reproductive structures develop on these gametophores, but they are unable to form any sporophyte, the only diploid stage in the moss life cycle. Finally, the mutant phenotypes of ΔPpXLG and ΔPpGβ2 can be complemented by the homologous genes from Arabidopsis, AtXLG2 and AtAGB1, respectively, suggesting an overall conservation of their function throughout the plant evolution.

Plastidial glycolipids contain diacylglycerol (DAG) moieties, which are either synthesized in the plastids (prokaryotic lipids) or originate in the extraplastidial compartment (eukaryotic lipids) necessitating their transfer into plastids. In contrast, the only phospholipid in plastids, phosphatidylglycerol (PG), contains exclusively prokaryotic DAG backbones. PG contributes in several ways to the functions of chloroplasts, but it is not known to what extent its prokaryotic nature is required to fulfill these tasks. As a first step toward answering this question, we produced transgenic tobacco plants that contain eukaryotic PG in thylakoids. This was achieved by targeting a bacterial DAG kinase into chloroplasts in which the heterologous enzyme was also incorporated into the envelope fraction. From lipid analysis we conclude that the DAG kinase phosphorylated eukaryotic DAG forming phosphatidic acid, which was converted into PG. This resulted in PG with 2-3 times more eukaryotic than prokaryotic DAG backbones. In the newly formed PG the unique Delta3-trans-double bond, normally confined to 3-trans-hexadecenoic acid, was also found in sn-2-bound cis-unsaturated C18 fatty acids. In addition, a lipidomics technique allowed the characterization of phosphatidic acid, which is assumed to be derived from eukaryotic DAG precursors in the chloroplasts of the transgenic plants. The differences in lipid composition had only minor effects on measured functions of the photosynthetic apparatus, whereas the most obvious phenotype was a significant reduction in growth.

Heterotrimeric G-proteins (G-proteins, hereafter) are important signaling components in all eukaryotes. The absence of these proteins in the sequenced genomes of Chlorophyaceaen green algae has raised questions about their evolutionary origin and prevalence in the plant lineage. The existence of G-proteins has often been correlated with the acquisition of embryophytic life-cycle and/or terrestrial habitats of plants which occurred around 450 million years ago. Our discovery of functional G-proteins in Chara braunii, a representative of the Charophycean green algae, establishes the existence of this conserved signaling pathway in the most basal plants and dates it even further back to 1-1.5 billion years ago. We have now identified the sequence homologs of G-proteins in additional algal families and propose that green algae represent a model system for one of the most basal forms of G-protein signaling known to exist to date. Given the possible differences that exist between plant and metazoan G-protein signaling mechanisms, such basal organisms will serve as important resources to trace the evolutionary origin of proposed mechanistic differences between the systems as well as their plant-specific functions.

Summary The influence of IAA, 2,4 D and tryptophan on bud formation was studied on whole protonema and on isolated caulonema filaments of Funaria hygrometrica. Whereas on whole protonema IAA, 2,4 D and tryptophan alone can enhance bud formation, on isolated filaments such an enhancement is only possible together with cytokinin. In this case kinetin and the auxin work synergistically. The antiauxin PCIB reverses the differentiation of caulonema to chloronema. Protonema which is treated with 3 × 10−4 M PCIB is not able to form buds either spontaneously or after treatment with kinetin. The IAA stimulated bud formation is inhibited by PCIB. Therefore it is assumed that a certain internal IAA level is important for caulonema differentiation.