A versatile CRISPR-based system for lineage tracing in living plants.

Individual cells give rise to diverse cell lineages during the development of multicellular organisms. Understanding the contribution of these lineages to mature organisms is a central question of developmental biology. Several techniques to document cell lineages have been used, from marking single cells with mutations that express a visible marker to generating molecular bar codes by CRISPR-induced mutations and subsequent single-cell analysis. Here, we exploit the mutagenic activity of CRISPR to allow lineage tracing within living plants with a single reporter. Cas9-induced mutations are directed to correct a frameshift mutation that restores expression of a nuclear fluorescent protein, labelling the initial cell and all progenitor cells with a strong signal without modifying other phenotypes of the plants. Spatial and temporal control of Cas9 activity can be achieved using tissue-specific and/or inducible promoters. We provide proof of principle for the function of lineage tracing in two model plants. The conserved features of the components and the versatile cloning system, allowing for easy exchange of promoters, are expected to make the system widely applicable.

Meristem dormancy in Marchantia polymorpha is regulated by a liverwort-specific miRNA and a clade III SPL gene.

The shape of modular organisms depends on the branching architecture, which in plants is determined by the fates of generative centers called meristems. The branches of the liverwort Marchantia polymorpha are derived from two adjacent meristems that develop at thallus apices. These meristems may be active and develop branches or may be dormant and do not form branches. The relative number and position of active and dormant meristems define the overall shape and form of the thallus. We show that the clade III SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factor MpSPL1 is required for meristem dormancy. The activity of MpSPL1 is regulated by the liverwort-specific Mpo-MR13 miRNA, which, in turn, is regulated by PIF-mediated signaling. An unrelated PIF-regulated miRNA, MIR156, represses a different SPL gene (belonging to clade IV) that inhibits branching during the shade avoidance response in Arabidopsis thaliana. This suggests that a conserved light signaling mechanism modulates branching architecture in liverworts and angiosperms and therefore is likely operated in the last common ancestor. However, PIF-mediated signaling represses the expression of different miRNA genes with different SPL targets during dichotomous, apical branching in liverworts and during lateral, subapical branching in angiosperms. We speculate that the mechanism that acts downstream of light and regulates meristem dormancy evolved independently in liverworts and angiosperms.

An Evolutionarily Conserved Receptor-like Kinases Signaling Module Controls Cell Wall Integrity During Tip Growth.

Rooting cells and pollen tubes-key adaptative innovations that evolved during the colonization and subsequent radiation of plants on land-expand by tip growth. Tip growth relies on a tight coordination between the protoplast growth and the synthesis/remodeling of the external cell wall. In root hairs and pollen tubes of the seed plant Arabidopsis thaliana, cell wall integrity (CWI) mechanisms monitor this coordination through the Malectin-like receptor kinases (MLRs), such as AtANXUR1 and AtFERONIA, that act upstream of the AtMARIS PTI1-like kinase. Here, we show that rhizoid growth in the early diverging plant, Marchantia polymorpha, is also controlled by an MLR and PTI1-like signaling module. Rhizoids, root hairs, and pollen tubes respond similarly to disruption of MLR and PTI1-like encoding genes. Thus, the MLR and PTI1-like signaling module that controls CWI during tip growth is conserved between M. polymorpha and A. thaliana, suggesting that it was active in the common ancestor of land plants.

Successive duplication-divergence mechanisms at the RCO locus contributed to leaf shape diversity in the Brassicaceae.

Gene duplication is a major driver for the increase of biological complexity. The divergence of newly duplicated paralogs may allow novel functions to evolve, while maintaining the ancestral one. Alternatively, partitioning the ancestral function among paralogs may allow parts of that role to follow independent evolutionary trajectories. We studied the REDUCED COMPLEXITY (RCO) locus, which contains three paralogs that have evolved through two independent events of gene duplication, and which underlies repeated events of leaf shape evolution within the Brassicaceae. In particular, we took advantage of the presence of three potentially functional paralogs in Capsella to investigate the extent of functional divergence among them. We demonstrate that the RCO copies control growth in different areas of the leaf. Consequently, the copies that are retained active in the different Brassicaceae lineages contribute to define the leaf dissection pattern. Our results further illustrate how successive gene duplication events and subsequent functional divergence can increase trait evolvability by providing independent evolutionary trajectories to specialized functions that have an additive effect on a given trait.

Diversification of a Transcription Factor Family Led to the Evolution of Antagonistically Acting Genetic Regulators of Root Hair Growth.

Streptophytes colonized the land some time before 470 million years ago [1-3]. The colonization coincided with an increase in morphological and cellular diversity [4-7]. This increase in diversity is correlated with a proliferation in transcription factors encoded in genomes [8-10]. This suggests that gene duplication and subsequent diversification of function was instrumental in the generation of land plant diversity. Here, we investigate the diversification of the streptophyte-specific Lotus japonicus ROOTHAIRLESS LIKE (LRL) transcription factor (TF) [11, 12] subfamily of basic loop helix (bHLH) proteins by comparing gene function in early divergent and derived land plant species. We report that the single Marchantia polymorpha LRL gene acts as a general growth regulator required for rhizoid development, a function that has been partially conserved throughout multicellular streptophytes. In contrast, the five relatively derived Arabidopsis thaliana LRL genes comprise two antagonistically acting groups of differentially expressed genes. The diversification of LRL genes accompanied the evolution of an antagonistic regulatory element controlling root hair development.