Convergent evolution of water conducting cells in Marchantia recruited the ZHOUPI gene promoting cell wall reinforcement and programmed cell death

A key adaptation of plants to life on land is the formation of water conducting cells (WCC) for efficient long-distance water transport. Based on morphological analyses it is thought that WCC have evolved independently on multiple occasions. For example, WCC have been lost in all but a few lineages of bryophytes but strikingly, within the liverworts a derived group, the complex thalloids, has evolved a novel externalised water conducting tissue composed of reinforced, hollow cells termed pegged rhizoids. Here we show that pegged rhizoid differentiation in Marchantia polymorpha is controlled by orthologues of the ZHOUPI and ICE bHLH transcription factors required for endosperm cell death in Arabidopsis seeds. By contrast, pegged rhizoid development was not affected by disruption of MpNAC5, the Marchantia orthologue of the VND genes that control WCC formation in flowering plants. We characterize the rapid, genetically controlled programmed cell death process that pegged rhizoids undergo to terminate cellular differentiation, and identify a corresponding upregulation of conserved putative plant cell death effector genes. Lastly, we show that ectopic expression of MpZOU1 increases production of pegged rhizoids and enhances drought tolerance. Our results support that pegged rhizoids having evolved independently of other WCC. We suggest that elements of the genetic control of developmental cell death are conserved throughout land plants and that the ZHOUPI/ICE regulatory module has been independently recruited to promote cell wall modification and programmed cell death in liverwort rhizoids and in the endosperm of flowering plant seed

Comparative analysis of the function and regulation of the ZOU/ICE protein complex in Arabidopsis thaliana and Marchantia polymorpha

In angiosperms, the development of the seed and the accumulation of reserves require the establishment of close communication between the embryo and the endosperm. In Arabidopsis thaliana, this coordination is regulated by a protein complex made up of two transcription factors: ZHOUPI and INDUCER OF CBF EXPRESSION (ZOU/ICE). The ZOU/ICE complex controls the degradation of the endosperm and thus the optimal development of the embryo, and regulates the establishment of a complex interface between the endosperm and the embryo composed of an extracuticular sheath and of the embryonic cuticle. In a first part of my work, the analysis of the regulation and the function of the ZOU/ICE complex made it possible to highlight the regulation underlying a cryptic redundancy in seed development between the two ICE genes in Arabidopsis thaliana, as well as to explore the post-translational regulation of their proteins, in particular the potential role of ubiquitination in their degradation via the HOS1 protein. Secondly, genetic and molecular and cellular biology analyses in the bryophyte Marchantia polymorpha, which does not produce seeds, provided a better understanding of the ancestral function of the ZOU/ICE complex. Indeed, this study revealed two potential ancestral functions of this complex, firstly in the control of the development of pegged rhizoids involved in the transport of water and secondly in the biogenesis of the cuticle. The study of the composition of the cuticle of Marchantia polymorpha, and of the transcriptional targets of the ZOU/ICE complex involved in its establishment, made it possible to formulate innovative hypotheses about the importance of the modification of the composition of the wall, and the production of reactive oxygen species in plant cuticle biogenesis

Analyse comparative de la fonction et de la régulation du complexe protéique ZOU/ICE chez Arabidopsis thaliana et Marchantia polymorpha

In angiosperms, the development of the seed and the accumulation of reserves require the establishment of close communication between the embryo and the endosperm. In Arabidopsis thaliana, this coordination is regulated by a protein complex made up of two transcription factors: ZHOUPI and INDUCER OF CBF EXPRESSION (ZOU/ICE). The ZOU/ICE complex controls the degradation of the endosperm and thus the optimal development of the embryo, and regulates the establishment of a complex interface between the endosperm and the embryo composed of an extracuticular sheath and of the embryonic cuticle. In a first part of my work, the analysis of the regulation and the function of the ZOU/ICE complex made it possible to highlight the regulation underlying a cryptic redundancy in seed development between the two ICE genes in Arabidopsis thaliana, as well as to explore the post-translational regulation of their proteins, in particular the potential role of ubiquitination in their degradation via the HOS1 protein. Secondly, genetic and molecular and cellular biology analyses in the bryophyte Marchantia polymorpha, which does not produce seeds, provided a better understanding of the ancestral function of the ZOU/ICE complex. Indeed, this study revealed two potential ancestral functions of this complex, firstly in the control of the development of pegged rhizoids involved in the transport of water and secondly in the biogenesis of the cuticle. The study of the composition of the cuticle of Marchantia polymorpha, and of the transcriptional targets of the ZOU/ICE complex involved in its establishment, made it possible to formulate innovative hypotheses about the importance of the modification of the composition of the wall, and the production of reactive oxygen species in plant cuticle biogenesis.Chez les Angiospermes, le développement de la graine et l’accumulation des réserves imposent la mise en place d’une communication étroite entre l’embryon et l’albumen. Chez certaines Angiospermes, comme l’arabette des dames aussi appelée Arabidopsis thaliana, ce dialogue entre embryon et albumen est régulé par un complexe protéique composé de deux facteurs de transcription : ZHOUPI et INDUCER OF CBF EXPRESSION1 (ZOU/ICE). Plus précisément, ce complexe ZOU/ICE permet la dégradation de l’albumen et donc le bon développement de l’embryon et régule également la mise en place d’une interface complexe entre l’albumen et l’embryon composée d’une gaine extracuticulaire et de la cuticule embryonnaire. Dans une première partie, l’analyse de la régulation et de la fonction du complexe ZOU/ICE a permis de mettre en évidence une redondance fonctionnelle cryptique entre les gènes ICEs au cours du processus de développement de la graine mais aussi la régulation post traductionnelle de ces protéines ICEs, en particulier l’ubiquitination participant à leur dégradation par l’action de la protéine HOS1. Dans un deuxième temps, des approches de génétique, de biologie moléculaire et cellulaire chez la Bryophyte Marchantia polymorpha, dépourvue de graines et d’albumen, ont permis de mieux comprendre la fonction ancestrale du complexe ZOU/ICE. Ce complexe s’est révélé avoir un rôle clé d’une part dans le contrôle du développement des rhizoïdes, impliqués dans le transport de l’eau, et d’autre part dans la biogénèse de la cuticule. L’étude de la composition de la cuticule de M. polymorpha, et des cibles transcriptionnelles du complexe ZOU/ICE impliquées dans sa mise en place, ont permis d’émettre des hypothèses novatrices sur l’importance de la modification de la composition de la paroi, et la production des espèces réactives de l’oxygène dans la biogenèse de la cuticule des plantes

Analyse comparative de la fonction et de la régulation du complexe protéique ZOU/ICE chez Arabidopsis thaliana et Marchantia polymorpha

Chez les Angiospermes, le développement de la graine et l’accumulation des réserves imposent la mise en place d’une communication étroite entre l’embryon et l’albumen. Chez certaines Angiospermes, comme l’arabette des dames aussi appelée Arabidopsis thaliana, ce dialogue entre embryon et albumen est régulé par un complexe protéique composé de deux facteurs de transcription : ZHOUPI et INDUCER OF CBF EXPRESSION1 (ZOU/ICE). Plus précisément, ce complexe ZOU/ICE permet la dégradation de l’albumen et donc le bon développement de l’embryon et régule également la mise en place d’une interface complexe entre l’albumen et l’embryon composée d’une gaine extracuticulaire et de la cuticule embryonnaire. Dans une première partie, l’analyse de la régulation et de la fonction du complexe ZOU/ICE a permis de mettre en évidence une redondance fonctionnelle cryptique entre les gènes ICEs au cours du processus de développement de la graine mais aussi la régulation post traductionnelle de ces protéines ICEs, en particulier l’ubiquitination participant à leur dégradation par l’action de la protéine HOS1. Dans un deuxième temps, des approches de génétique, de biologie moléculaire et cellulaire chez la Bryophyte Marchantia polymorpha, dépourvue de graines et d’albumen, ont permis de mieux comprendre la fonction ancestrale du complexe ZOU/ICE. Ce complexe s’est révélé avoir un rôle clé d’une part dans le contrôle du développement des rhizoïdes, impliqués dans le transport de l’eau, et d’autre part dans la biogénèse de la cuticule. L’étude de la composition de la cuticule de M. polymorpha, et des cibles transcriptionnelles du complexe ZOU/ICE impliquées dans sa mise en place, ont permis d’émettre des hypothèses novatrices sur l’importance de la modification de la composition de la paroi, et la production des espèces réactives de l’oxygène dans la biogenèse de la cuticule des plantes.In angiosperms, the development of the seed and the accumulation of reserves require the establishment of close communication between the embryo and the endosperm. In Arabidopsis thaliana, this coordination is regulated by a protein complex made up of two transcription factors: ZHOUPI and INDUCER OF CBF EXPRESSION (ZOU/ICE). The ZOU/ICE complex controls the degradation of the endosperm and thus the optimal development of the embryo, and regulates the establishment of a complex interface between the endosperm and the embryo composed of an extracuticular sheath and of the embryonic cuticle. In a first part of my work, the analysis of the regulation and the function of the ZOU/ICE complex made it possible to highlight the regulation underlying a cryptic redundancy in seed development between the two ICE genes in Arabidopsis thaliana, as well as to explore the post-translational regulation of their proteins, in particular the potential role of ubiquitination in their degradation via the HOS1 protein. Secondly, genetic and molecular and cellular biology analyses in the bryophyte Marchantia polymorpha, which does not produce seeds, provided a better understanding of the ancestral function of the ZOU/ICE complex. Indeed, this study revealed two potential ancestral functions of this complex, firstly in the control of the development of pegged rhizoids involved in the transport of water and secondly in the biogenesis of the cuticle. The study of the composition of the cuticle of Marchantia polymorpha, and of the transcriptional targets of the ZOU/ICE complex involved in its establishment, made it possible to formulate innovative hypotheses about the importance of the modification of the composition of the wall, and the production of reactive oxygen species in plant cuticle biogenesis

Analyse comparative de la fonction et de la régulation du complexe protéique ZOU/ICE chez Arabidopsis thaliana et Marchantia polymorpha

In angiosperms, the development of the seed and the accumulation of reserves require the establishment of close communication between the embryo and the endosperm. In Arabidopsis thaliana, this coordination is regulated by a protein complex made up of two transcription factors: ZHOUPI and INDUCER OF CBF EXPRESSION (ZOU/ICE). The ZOU/ICE complex controls the degradation of the endosperm and thus the optimal development of the embryo, and regulates the establishment of a complex interface between the endosperm and the embryo composed of an extracuticular sheath and of the embryonic cuticle. In a first part of my work, the analysis of the regulation and the function of the ZOU/ICE complex made it possible to highlight the regulation underlying a cryptic redundancy in seed development between the two ICE genes in Arabidopsis thaliana, as well as to explore the post-translational regulation of their proteins, in particular the potential role of ubiquitination in their degradation via the HOS1 protein. Secondly, genetic and molecular and cellular biology analyses in the bryophyte Marchantia polymorpha, which does not produce seeds, provided a better understanding of the ancestral function of the ZOU/ICE complex. Indeed, this study revealed two potential ancestral functions of this complex, firstly in the control of the development of pegged rhizoids involved in the transport of water and secondly in the biogenesis of the cuticle. The study of the composition of the cuticle of Marchantia polymorpha, and of the transcriptional targets of the ZOU/ICE complex involved in its establishment, made it possible to formulate innovative hypotheses about the importance of the modification of the composition of the wall, and the production of reactive oxygen species in plant cuticle biogenesis.Chez les Angiospermes, le développement de la graine et l’accumulation des réserves imposent la mise en place d’une communication étroite entre l’embryon et l’albumen. Chez certaines Angiospermes, comme l’arabette des dames aussi appelée Arabidopsis thaliana, ce dialogue entre embryon et albumen est régulé par un complexe protéique composé de deux facteurs de transcription : ZHOUPI et INDUCER OF CBF EXPRESSION1 (ZOU/ICE). Plus précisément, ce complexe ZOU/ICE permet la dégradation de l’albumen et donc le bon développement de l’embryon et régule également la mise en place d’une interface complexe entre l’albumen et l’embryon composée d’une gaine extracuticulaire et de la cuticule embryonnaire. Dans une première partie, l’analyse de la régulation et de la fonction du complexe ZOU/ICE a permis de mettre en évidence une redondance fonctionnelle cryptique entre les gènes ICEs au cours du processus de développement de la graine mais aussi la régulation post traductionnelle de ces protéines ICEs, en particulier l’ubiquitination participant à leur dégradation par l’action de la protéine HOS1. Dans un deuxième temps, des approches de génétique, de biologie moléculaire et cellulaire chez la Bryophyte Marchantia polymorpha, dépourvue de graines et d’albumen, ont permis de mieux comprendre la fonction ancestrale du complexe ZOU/ICE. Ce complexe s’est révélé avoir un rôle clé d’une part dans le contrôle du développement des rhizoïdes, impliqués dans le transport de l’eau, et d’autre part dans la biogénèse de la cuticule. L’étude de la composition de la cuticule de M. polymorpha, et des cibles transcriptionnelles du complexe ZOU/ICE impliquées dans sa mise en place, ont permis d’émettre des hypothèses novatrices sur l’importance de la modification de la composition de la paroi, et la production des espèces réactives de l’oxygène dans la biogenèse de la cuticule des plantes

Evaluation of genome and base editing tools in maize protoplasts

International audienceIntroduction Despite its rapid worldwide adoption as an efficient mutagenesis tool, plant genome editing remains a labor-intensive process requiring often several months of in vitro culture to obtain mutant plantlets. To avoid a waste in time and money and to test, in only a few days, the efficiency of molecular constructs or novel Cas9 variants (clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9) prior to stable transformation, rapid analysis tools are helpful. Methods To this end, a streamlined maize protoplast system for transient expression of CRISPR/Cas9 tools coupled to NGS (next generation sequencing) analysis and a novel bioinformatics pipeline was established. Results and discussion Mutation types found with high frequency in maize leaf protoplasts had a trend to be the ones observed after stable transformation of immature maize embryos. The protoplast system also allowed to conclude that modifications of the sgRNA (single guide RNA) scaffold leave little room for improvement, that relaxed PAM (protospacer adjacent motif) sites increase the choice of target sites for genome editing, albeit with decreased frequency, and that efficient base editing in maize could be achieved for certain but not all target sites. Phenotypic analysis of base edited mutant maize plants demonstrated that the introduction of a stop codon but not the mutation of a serine predicted to be phosphorylated in the bHLH (basic helix loop helix) transcription factor ZmICEa (INDUCER OF CBF EXPRESSIONa) caused abnormal stomata, pale leaves and eventual plant death two months after sowing

Stomatal regulators are co-opted for seta development in the astomatous liverwort Marchantia polymorpha

International audienceThe evolution of special types of cells requires the acquisition of new gene regulatory networks controlled by transcription factors (TFs). In stomatous plants, a TF module formed by subfamilies Ia and IIIb basic helix-loop-helix TFs (Ia-IIIb bHLH) regulates stomatal formation; however, how this module evolved during land plant diversification remains unclear. Here we show that, in the astomatous liverwort Marchantia polymorpha, a Ia-IIIb bHLH module regulates the development of a unique sporophyte tissue, the seta, which is found in mosses and liverworts. The sole Ia bHLH gene, MpSETA, and a IIIb bHLH gene, MpICE2, regulate the cell division and/or differentiation of seta lineage cells. MpSETA can partially replace the stomatal function of Ia bHLH TFs in Arabidopsis thaliana, suggesting that a common regulatory mechanism underlies setal and stomatal formation. Our findings reveal the co-option of a Ia-IIIb bHLH TF module for regulating cell fate determination and/or cell division of distinct types of cells during land plant evolution.In land plants, the Ia and IIIb basic helix-loop-helix (Ia-IIIb bHLH) module regulates stomatal development. This study shows that a Ia-IIIb bHLH heterodimer, MpSETA-MpICE2, regulates seta formation in Marchantia and suggests a common mechanism underlying stomata and setae formation

Stomatal regulators are co-opted for seta development in the astomatous liverwort Marchantia polymorpha

The evolution of special types of cells requires the acquisition of new gene regulatory networks controlled by transcription factors (TFs). In stomatous plants, a TF module formed by subfamilies Ia and IIIb basic helix-loop-helix TFs (Ia-IIIb bHLH) regulates stomatal formation; however, how this module evolved during land plant diversification remains unclear. Here, we show that, in the astomatous liverwort Marchantia polymorpha , a Ia-IIIb bHLH module regulates the development of a unique sporophyte tissue, the seta, which is found in mosses and liverworts. The sole Ia bHLH gene, Mp SETA , and a IIIb bHLH gene, Mp ICE2 , regulate the cell division and/or differentiation of seta lineage cells. MpSETA can partially replace the stomatal function of Ia bHLH TFs in Arabidopsis thaliana , suggesting that a common regulatory mechanism underlies the setal and stomatal formation. Our findings reveal the co-option of a Ia-IIIb bHLH TF module for regulating cell fate determination and/or cell division of distinct types of cells during land plant evolution

The LEAFY floral regulator displays pioneer transcription factor properties

International audiencePioneer transcription factors (TFs) are a special category of TFs with the capacity to bind to closed chromatin regions in which DNA is wrapped around histones and may be highly methylated. Subsequently, pioneer TFs are able to modify the chromatin state to initiate gene expression. In plants, LEAFY (LFY) is a master floral regulator and has been suggested to act as a pioneer TF in Arabidopsis. Here, we demonstrate that LFY is able to bind both methylated and non-methylated DNA using a combination of in vitro genome-wide binding experiments and structural modeling. Comparisons between regions bound by LFY in vivo and chromatin accessibility data suggest that a subset of LFY bound regions is occupied by nucleosomes. We confirm that LFY is able to bind nucleosomal DNA in vitro using reconstituted nucleosomes. Finally, we show that constitutive LFY expression in seedling tissues is sufficient to induce chromatin accessibility in the LFY direct target genes, APETALA1 and AGAMOUS. Taken together, our study suggests that LFY possesses key pioneer TF features that contribute to launch the floral gene expression program