Patrice Morel

Fruit development is a key angiosperm innovation for seed dispersal, yet it remains unclear whether a conserved gene regulatory network operates during early fruit development. We investigated Petunia × hybrida homologs of FRUITFULL ( FUL ), SHATTERPROOF ( SHP ), and APETALA 2 ( AP2 ), transcription factors repeatedly linked to fruit development/ripening. As a model species close to tomato, but producing dry dehiscent fruits like Arabidopsis, petunia is ideal for investigating conserved regulatory mechanisms in early fruit patterning. We used reverse genetics to dissect the roles of the petunia FUL , SHP and AP2 homologs in pericarp development. We found that the three petunia FUL -like genes redundantly regulate endocarp formation, likely via auxin/brassinosteroid signalling and cell-wall modification. Interestingly, FBP6 ( SHP-like ) has an opposite effect on endocarp cell morphology, suggesting antagonistic interactions. Mutants in the AP2- like ROB genes show the most severe pericarp patterning defects, with failure of dehiscence in rob1/2/3 mutants . ROB factors repress FUL -like activity and, along with FBP6, oppositely regulate auxin/brassinosteroid signalling genes. Our findings uncover a conserved module of antagonistically acting transcription factors (AP2, FUL, SHP co-orthologs) that regulates early pericarp patterning via hormonal signalling, providing a framework for comparative fruit development studies across angiosperms. Highlight: Three key transcription factors in plant reproductive development, FRUITFULL, SHATTERPROOF and APETALA 2, regulate petunia fruit development and together control pericarp patterning in the young fruit.

In flowering plants, floral organ identity is specified by the combinatorial action of homeotic genes. While the role of these genes in the early specification of organ identity is well established, their late function throughout floral organ development and in specific cell types is much less characterized. In particular, since plant organs are structured in clonally-independent cell layers, whether and how homeotic identity interacts with cell layer identity is unknown. We have previously identified cell layer-specific mutants for the petal identity gene PhDEF in petunia flowers, resulting in drastically different petal phenotypes whether PhDEF is expressed in the petal epidermis or in the mesophyll. In this study, using a combination of single-cell RNA-Seq and chromatin immunoprecipitation on phdef cell layer-specific mutants, we find that PhDEF regulates a different set of target genes in the petal epidermis and mesophyll, with a major regulatory action in the epidermis. We uncover a high diversity of binding profiles in PhDEF target genes, with a complex combination of layer-specific or -aspecific binding sites, and a much more prominent binding of PhDEF in the epidermis than in the mesophyll. Our study highlights that floral homeotic genes like PhDEF can have different regulatory actions in different cell contexts, here different cell layers, and that cell layer identity indeed influences the regulatory processes underlying homeotic identity.

Plants have colonized lands 450 million years ago. This terrestrialization was facilitated by developmental and functional innovations. Recent evo-devo approaches have demonstrated that one of these innovations was the mutualistic arbuscular mycorrhizal symbiosis (AMS). The genetic pathways that have been involved in the establishment and functioning of AMS since its evolution remain poorly described. Here, we found that intracellular colonization by AM fungi induces a transcriptional reporter of the common symbiosis pathway, well-described in angiosperms, in the liverwort Marchantia paleacea . Mutants of either of the three main genes of this pathway, SYMRK , CCaMK, and CYCLOPS, disrupt the ability of M. paleacea to associate with AM fungi. Finally, overexpressing gain-of-function CCaMK or CYCLOPS leads to convergent transcriptomic signatures that partially overlap with AMS. Altogether, our data indicate that plants have maintained three genes of the common symbiotic pathway to support symbiotic interactions since their most recent common ancestor.

Summary: Highlight: The common symbiotic pathway is activated during arbuscular mycorrhizal symbiosis in Marchantia paleacea The three core members of the common symbiotic pathway are essential for symbiosis in Marchantia paleacea The molecular function of the CCaMK/CYCLOPS module is conserved across land plants Symbiotic signalling has been conserved in plants for 450 million years The colonization of land by plants 450 million years ago revolutionized life on Earth ¹ . The fossil record ² and genetic evidence in extant species ³ suggest that this transition was facilitated by interactions with symbiotic arbuscular mycorrhizal (AM) fungi ⁴ . This ancestral symbiosis relied on the biosynthesis of chemicals by the host plant, both as signals ⁵ and as nutrients ³ . In angiosperms, a signalling pathway involving the receptor-like kinase SYMRK/DMI2 ^6,7 , the Calcium and Calmodulin-dependent protein kinase CCaMK/DMI3 ⁸ and the transcription factor CYCLOPS/IPD3 ^9,10 has been described as the common symbiosis pathway (CSP), essential for the establishment of the AM symbiosis and the root-nodule symbiosis ¹¹ . Phylogenetic and comparative phylogenomic analyses indicated an ancient origin of the CSP, present in all extant land plants forming intracellular symbioses ^12–15 . Trans-complementation assays of the angiosperm mutants with orthologs from diverse species further indicated the conservation of the molecular function of the CSP across the embryophytes ^9,12,14–16 . However, this correlative evidence did not allow testing the ancestral biological function of the CSP. In this study we demonstrate that SYMRK, CCaMK and CYCLOPS are essential for the colonization by AM fungi in bryophytes, indicating that plants have maintained a dedicated signalling pathway to support symbiotic interactions for 450 million years.

Floral homeotic MADS-box transcription factors ensure the correct morphogenesis of floral organs, which are organized in different cell layers deriving from distinct meristematic layers. How cells from these distinct layers acquire their respective identities and coordinate their growth to ensure normal floral organ morphogenesis is unresolved. Here, we studied petunia (Petunia × hybrida) petals that form a limb and tube through congenital fusion. We identified petunia mutants (periclinal chimeras) expressing the B-class MADS-box gene DEFICIENS in the petal epidermis or in the petal mesophyll, called wico and star, respectively. Strikingly, wico flowers form a strongly reduced tube while their limbs are almost normal, while star flowers form a normal tube but greatly reduced and unpigmented limbs, showing that petunia petal morphogenesis is highly modular. These mutants highlight the layer-specific roles of PhDEF during petal development. We explored the link between PhDEF and petal pigmentation, a well-characterized limb epidermal trait. The anthocyanin biosynthesis pathway was strongly downregulated in star petals, including its major regulator ANTHOCYANIN2 (AN2). We established that PhDEF directly binds to the AN2 terminator in vitro and in vivo, suggesting that PhDEF might regulate AN2 expression and therefore petal epidermis pigmentation. Altogether, we show that cell layer–specific homeotic activity in petunia petals differently impacts tube and limb development, revealing the relative importance of the different cell layers in the modular architecture of petunia petals.

Functional analysis of the petunia MADS-box gene SEP/AGL6/AP1 superclade compared to Arabidopsis and other species suggests major differences in the functional

The nucleoplasm and nucleolus are the two main territories of the nucleus. While specific functions are associated with each of these territories (such as mRNA synthesis in the nucleoplasm and ribosomal rRNA synthesis in the nucleolus), some proteins are known to be located in both. Here, we investigated the molecular function of REBELOTE (RBL), an Arabidopsis thaliana protein previously characterized as a regulator of floral meristem termination. We show that RBL displays a dual localization, in the nucleolus and nucleoplasm. Moreover, we used direct and global approaches to demonstrate that RBL interacts with nucleic acid‐binding proteins. It binds to the NOC proteins SWA2, AtNOC2 and AtNOC3 in both the nucleolus and nucleoplasm, and also to OBE1 and VFP3/ENAP1. Taking into account the identities of these RBL interactors, we hypothesise that RBL acts both in ribosomal biogenesis and in the regulation of gene expression.

To attract insects, flowers produce nectar, an energy-rich substance secreted by specialized organs called nectaries. For Arabidopsis thaliana, a rosid species with stamen-associated nectaries, the floral B-, C-, and E-functions were proposed to redundantly regulate nectary development. Here, we investigated the molecular basis of carpel-associated nectary development in the asterid species petunia (Petunia hybrida). We show that its euAGAMOUS (euAG) and PLENA (PLE) C-lineage MADS box proteins are essential for nectary development, while their overexpression is sufficient to induce ectopic nectaries on sepals. Furthermore, we demonstrate that Arabidopsis nectary development also fully depends on euAG/PLE C-lineage genes. In turn, we show that petunia nectary development depends on two homologs of CRABS CLAW (CRC), a gene previously shown to be required for Arabidopsis nectary development, and demonstrate that CRC expression in both species depends on the members of both euAG/PLE C-sublineages. Therefore, petunia and Arabidopsis employ a similar molecular mechanism underlying nectary development, despite otherwise major differences in the evolutionary trajectory of their C-lineage genes, their distant phylogeny, and different nectary positioning. However, unlike in Arabidopsis, petunia nectary development is position independent within the flower. Finally, we show that the TARGET OF EAT-type BLIND ENHANCER and APETALA2-type REPRESSOR OF B-FUNCTION genes act as major regulators of nectary size.