The evolutionary success of flowering plants is largely attributed to the development of protective maternal tissues such as the fruit and seed coat, which facilitate seed dispersal. However, the precise mechanisms by which fertilization triggers the development of these specialized maternal tissues remain poorly understood. A critical event in this process is the initiation of auxin biosynthesis within the endosperm, a tissue that subsequently signals to the surrounding maternal tissues (seed coat and fruit) to promote their development. Previous research using strawberry, *Fragaria vesca*, highlighted the importance of auxin as an inductive signal originating from the seed, as removal of achenes (seed-containing ovaries) prevents fruit flesh development, while exogenous auxin application can stimulate fruit growth in the absence of achenes. Genetic studies in Arabidopsis and tomato further support the conserved role of auxin in fruit development, with mutants exhibiting defects in auxin signaling often producing parthenocarpic (seedless) fruits. Transcriptomic studies in *F. vesca* have shown that the expression of auxin biosynthesis genes (YUCs and TAAs) is predominantly induced in the endosperm post-fertilization. Despite these advancements, the molecular mechanisms regulating this fertilization-induced auxin synthesis in the endosperm remain elusive. The endosperm is not solely a nutritive tissue; it functions as a 'sensor' coordinating embryo development with that of surrounding maternal tissues. Studies manipulating auxin biosynthesis in the Arabidopsis central cell (which gives rise to the endosperm) have demonstrated the importance of auxin in endosperm proliferation, seed coat differentiation, and fruit development. The timing of endosperm cellularization is also critical, as it determines the extent of nuclear proliferation and thus seed size. Auxin levels appear to regulate this process, with increased auxin preventing cellularization and leading to seed arrest. Therefore, understanding the control of auxin biosynthesis within the endosperm is crucial for improving grain and fruit yields. The type I MADS-box gene *AtAGL62* in Arabidopsis has been implicated in endosperm cellularization, with *atagl62* mutants showing precocious cellularization. While previous work suggested *AtAGL62* regulates the expression of an ABCB-type auxin transporter involved in transport from the endosperm to the seed coat, the relationship between *AtAGL62* and auxin biosynthesis remained unclear. This research investigates the mechanisms underlying fertilization-induced auxin biosynthesis in the endosperm, using the diploid wild strawberry *F. vesca* as a model to understand how fertilization stimulates fleshy fruit initiation. The researchers hypothesized that *FveAGL62* plays a pivotal role in this process, and that this mechanism is evolutionarily conserved in flowering plants.

Extensive research has established the critical role of auxin in fruit development. Early work by Nitsch (1950) demonstrated the effect of auxin on strawberry growth and morphogenesis. Subsequent studies in Arabidopsis and tomato have identified multiple auxin-related genes and pathways involved in fruit set and development. For instance, mutations in auxin signaling components often lead to parthenocarpic fruit development (Wang et al., 2005; Goetz et al., 2006, 2007; de Jong et al., 2009, 2015; Joldersma & Liu, 2018). These findings highlight the conserved nature of auxin-dependent mechanisms across various flowering plant species. Genome-wide studies, particularly transcriptomic analyses in strawberry (Kang et al., 2013), have revealed the spatial and temporal expression patterns of auxin biosynthesis genes in developing seeds and fruits, emphasizing the endosperm's crucial role as the primary site of fertilization-induced auxin production. The endosperm's role in coordinating embryo and maternal tissue development has been further explored (Costa et al., 2004), with studies showing that manipulating auxin biosynthesis in the endosperm directly affects seed and fruit development (Figueiredo et al., 2015, 2016). These findings established auxin as a key signal mediating the communication between the developing embryo and maternal tissues, highlighting the endosperm's role as a critical signaling center. The regulation of endosperm cellularization, a key developmental transition that influences seed size, has also been linked to auxin levels (Batista et al., 2019). Previous work on the Arabidopsis *AtAGL62* gene highlighted its involvement in regulating endosperm cellularization (Kang et al., 2008; Roszak & Köhler, 2011; Hehenberger et al., 2012; Xu et al., 2016), but its functional relationship with auxin biosynthesis was yet to be established. This previous research set the stage for the current study, which aims to elucidate the detailed molecular mechanism linking fertilization, *AGL62* function, and auxin biosynthesis in the endosperm, providing a deeper understanding of this critical developmental process.

This study employed a multifaceted approach integrating genetic analysis, transcriptomics, confocal microscopy, hormone treatments, and molecular techniques to investigate the role of *FveAGL62* in fertilization-induced auxin synthesis. The researchers utilized the diploid wild strawberry *Fragaria vesca* as a model system due to its readily accessible achenes and its established use in studying fruit development. Co-expression network analysis of previously generated transcriptomic data identified *FveAGL62* as a candidate gene for further investigation. CRISPR/Cas9 technology was used to generate *fveagl62* knockout mutants in *F. vesca*, allowing the assessment of the gene's function in seed and fruit development. The phenotypic characterization of these mutants included assessing fruit development, seed viability, and endosperm morphology. Reporter gene analysis (*ProFveAGL62::FveAGL62-GUS*) was used to confirm the spatial and temporal expression pattern of *FveAGL62*. To determine the mode of inheritance of *FveAGL62*, reciprocal crosses between wild-type and *fveagl62* plants were conducted. Hormone treatments (GA3 and NAA) were applied to *fveagl62* mutants to assess the involvement of auxin and gibberellins in the observed phenotypes. RNA sequencing (RNA-seq) was performed on stage 2 seeds from wild-type and *fveagl62* mutants to identify downstream genes and pathways regulated by *FveAGL62*. Differential gene expression analysis was conducted to identify genes exhibiting altered expression in the *fveagl62* mutants. Gene Ontology (GO) enrichment analysis was used to characterize the biological processes and pathways associated with the differentially expressed genes. RT-qPCR was used to validate RNA-seq results for selected genes, including auxin and gibberellin biosynthesis genes. Confocal laser scanning microscopy (CLSM) was employed to examine the endosperm morphology in detail, including assessing the timing of cellularization. To further investigate the role of *AGL62* and its conservation across species, Arabidopsis *atagl62* mutants were analyzed using similar techniques. Auxin reporters (*R2D2*, *ProAtYUC10::3xnGFP*) were used to measure auxin levels in *atagl62* mutants. Exogenous auxin application was used to rescue the phenotype in Arabidopsis mutants. Yeast one-hybrid (Y1H) assays were used to investigate protein-promoter interactions, including the binding of *FveAGL62* and *FveAGL80* to the promoters of auxin biosynthesis genes and *FveATHB* genes. Yeast two-hybrid (Y2H) assays and bimolecular fluorescence complementation (BiFC) assays were utilized to study protein-protein interactions between *FveAGL62*, *FveAGL80*, and other *FveATHB* genes. Transient luciferase assays in tobacco leaves were used to investigate the effect of *FveAGL62*, *FveAGL80*, and *FveATHB* genes on gene expression. Finally, overexpression lines of *FveATHB29b* and *FveATHB30* were generated to investigate their roles in auxin biosynthesis and fruit development. The methodologies involved a combination of molecular biology, genetics, microscopy, and bioinformatics approaches to comprehensively investigate the role of *AGL62* in auxin regulation during seed and fruit development.

This research identified *FveAGL62*, a type I MADS-box gene, as a crucial regulator of fertilization-induced auxin biosynthesis in the endosperm. The study demonstrated that *FveAGL62* is essential for normal seed and fruit development in *F. vesca*. *fveagl62* mutant plants showed severely impaired fruit development, producing small, non-viable seeds. The *FveAGL62* gene is specifically and transiently expressed in the endosperm immediately after fertilization. The study determined that *FveAGL62* does not undergo parental imprinting. RNA-seq analysis of *fveagl62* mutant seeds revealed that several auxin biosynthesis genes (*FveYUC1*, *FveYUC5*, *FveYUC10*, *FveTAA1*, *FveTAR1*, and *FveTAR2*) showed significantly reduced expression, confirming that *FveAGL62* positively regulates auxin biosynthesis. Gibberellin biosynthesis genes were also found to be downregulated in the mutants. Furthermore, the study showed that *FveAGL62* mutants exhibited premature endosperm cellularization, a phenotype partially rescued by exogenous auxin treatment. The findings are supported by similar experiments in Arabidopsis where *AtAGL62* also plays a role in auxin biosynthesis in the endosperm. The researchers discovered a regulatory module involving *FveAGL62/FveAGL80* and *FveATHB* transcription factors. The study showed that *FveAGL62/FveAGL80* directly repress the expression of *FveATHB29b* and *FveATHB30*, which in turn repress the expression of auxin biosynthesis genes like *FveYUC10*. Overexpression of *FveATHB29b* and *FveATHB30* resulted in smaller fruits and reduced auxin biosynthesis, consistent with their role as negative regulators of auxin synthesis. In summary, the data strongly support a conserved role for AGL62 in positively regulating auxin biosynthesis in the endosperm of diverse flowering plants. The study also noted that unlike in Arabidopsis, *FveAGL62* does not appear to be essential for seed coat development in strawberry. The results are summarized in a proposed model that outlines how fertilization, *FveAGL62/FveAGL80* expression, and *FveATHB* repression coordinate auxin biosynthesis to stimulate fruit development.

This research provides compelling evidence for a conserved mechanism regulating fertilization-induced auxin biosynthesis in the endosperm. The findings establish a crucial role for the type I MADS-box gene *AGL62* in promoting auxin synthesis, a process essential for successful seed and fruit development. The remarkable conservation of this mechanism between strawberry and Arabidopsis underscores its fundamental importance in flowering plant reproduction. The identification of *FveATHB* genes as downstream targets of *FveAGL62/FveAGL80*, functioning as negative regulators of auxin biosynthesis, reveals a sophisticated regulatory network controlling auxin levels in the endosperm. The observation that exogenous auxin partially rescues the phenotypes of *fveagl62* and *atagl62* mutants strengthens the causal link between *AGL62* function and auxin levels. The distinct roles of *AGL62* in seed coat development between strawberry and Arabidopsis suggest species-specific adaptations in reproductive strategies, highlighting the complexity and diversity of developmental processes within flowering plants. This work has significant implications for crop improvement, as understanding the regulatory mechanisms of auxin biosynthesis could provide targets for manipulating seed and fruit size. The research also opens new avenues for studying the evolution of reproductive processes and developing strategies for parthenocarpic fruit production. However, further research is required to fully elucidate the mechanisms underlying *AGL62* regulation and the interactions between different regulatory components of this network.

This study reveals a key molecular mechanism controlling fertilization-induced auxin synthesis in the endosperm, a process critical for seed and fruit development in flowering plants. The type I MADS-box gene *AGL62* plays a conserved and essential role in this process, acting through a regulatory network involving *AGL80* and *ATHB* transcription factors. This research provides crucial insights into plant reproductive biology and offers potential targets for crop improvement strategies aimed at increasing yield and manipulating fruit development. Future studies should focus on elucidating the upstream regulatory mechanisms controlling *AGL62* expression and further dissecting the interactions within this complex regulatory network.

While the study provides strong evidence for the role of *AGL62* in auxin biosynthesis, it doesn't fully elucidate the upstream regulatory mechanisms controlling *AGL62* expression. The precise mechanisms by which fertilization triggers *AGL62* expression remain to be determined. The study mainly focused on two model species, strawberry and Arabidopsis; further investigations are necessary to determine the extent of conservation and species-specific variations in this regulatory mechanism across a broader range of flowering plants. Finally, while exogenous auxin partially rescued some mutant phenotypes, it didn't fully restore normal development, indicating additional factors are involved. The overexpression experiments, using a constitutive promoter, may have introduced ectopic expression of *FveATHB* genes, potentially influencing other developmental processes and obscuring the direct effects on endosperm development.