SPOROCYTELESS/NOZZLE cooperates with MADS-domain transcription factors to regulate an auxin-dependent network controlling Megaspore-Mother-Cell differentiation

Domain: biology
Published: 2025-12-14
Editorial outcome: major_revision

Abstract
The formation of the female gamete is a complex developmental process that begins with the differentiation of the Megaspore Mother Cell (MMC) within the ovule. SPOROCYTELESS/NOZZLE (SPL/NZZ) is the principal regulator of the MMC formation, as mutations in the SPL/NZZ gene lead to the failure of the MMC differentiation. Nonetheless, the SPL/NZZ-dependent regulatory pathway governing the MMC development remains largely unknown. Using a multi-omics approach, we identify direct SPL/NZZ targets and their downstream network. We discover that SPL/NZZ interacts with ovule-identity MADS-domain transcription factor complexes to regulate the expression of common target genes. By integrating the omics data with the analysis of either complementation or mutant lines, we describe a comprehensive regulatory mechanism in which SPL/NZZ controls the differentiation of the MMC by acting on an auxin-dependent downstream network.

Introduction
In flowering plants, sporogenesis starts with the differentiation of female and male meiocytes: Megaspore Mother Cells (MMCs) within ovules and Pollen Mother Cells (PMCs) within anthers1,2. In Arabidopsis thaliana, the master regulator of meiocytes differentiation is SPOROCYTELESS/NOZZLE (SPL/NZZ), since mutations in the SPL/NZZ gene result in complete female and male sterility due to the absence of both the MMC and the PMC1,2. SPL/NZZ belongs to the SPEAR family, which consists of 5 genes3,4 in A. thaliana. The SPEAR proteins have a nuclear localisation signal at the N-terminus, the SPL motif in the middle of the protein, and an EAR motif at the C-terminus. It has been reported that, through the EAR motif, SPL/NZZ recruits co-repressors, like TOPLESS (TPL), that mediate the transcriptional repression of target genes by interacting with histone deacetylases (HDAs)3–7.

SPL/NZZ also has an additional Myc-type helix-loop-helix domain that could mediate SPL/NZZ homodimerization2,4. Interestingly, SPL/NZZ was initially described as a MADS-like protein, since it contains a sequence of 17 amino acids similar to the MADS-domain2.

According to earlier reports, SPL/NZZ can interact with two families of transcription factors (TFs): the YABBY (YAB) and the CINCINNATA-like TEOSINTE BRANCHED/CYCLOIDEA/PCF (TCP). SPL/NZZ interaction with these TFs mediates the transcriptional repression of their targets4,5,8.

Despite its importance for plant reproduction, the mechanisms of SPL/NZZ transcriptional regulation, as well as its downstream network, are still poorly understood. Among the factors involved in SPL/NZZ regulation, auxin signalling was shown to be a positive regulator of SPL/NZZ expression in anthers, as demonstrated by the reduced SPL/NZZ transcript accumulation in the taa1 tar2-2 mutant9. AGAMOUS (AG) is one of the few factors described to regulate SPL/NZZ expression in both ovules and stamens. Indeed, in flowers at stages 11–1310, AG binds a CArG-box in the SPL/NZZ 3′ UTR to promote its expression in integuments and chalaza of the ovule. Here, the SPL/NZZ expression pattern is also regulated by the RdDM pathway, since SPL/NZZ is ectopically expressed in the drm1 drm2 mutant11.

In addition to its function in MMC and PMC differentiation, SPL/NZZ is required for ovule patterning by negatively regulating the expression of factors such as AINTEGUMENTA (ANT), BELL1 (BEL1), INNER-NO-OUTER (INO), and PHABULOSA (PHB)12,13. Indeed, besides the absence of the MMC, spl/nzz mutant ovules are characterised by an impairment in integuments growth and a longer funiculus12.

Concerning the SPL/NZZ downstream network, SPL/NZZ plays a crucial role in the auxin signalling pathway, as the auxin-response reporter line DR5v2 and the auxin efflux carrier PIN-FORMED1 (PIN1) exhibit decreased expression in the spl/nzz mutant ovule14,15.

This study employs a multi-techniques approach, integrating Co-immunoprecipitation coupled with mass spectrometry (Co-IP/MS), Chromatin Immunoprecipitation and sequencing (ChIP-seq), and RNA sequencing (RNA-seq), to comprehensively identify direct SPL/NZZ targets and their downstream network. Our findings reveal the interaction between SPL/NZZ and ovule identity MADS-domain transcription factor complexes, thereby regulating the expression of common target genes, such as ANT. This activity influences the auxin signalling, which was previously suggested as a primary pathway necessary for the MMC differentiation15. We propose a multistep model in which MMC development relies on an SPL/NZZ-dependent auxin downstream network.

Results
SPL/NZZ and MADS-domain transcription factors interact to form multimeric complexes

To gain insight into the network regulated by SPL/NZZ, essential for the differentiation of the MMC, we identified potential SPL/NZZ partners during early stages of ovule development, before and at the MMC stage, by performing co-immunoprecipitation followed by mass spectrometry (Co-IP/MS). To do this, we took advantage of the pSPL/NZZ::SPL/NZZ:GFP construct, previously used to complement the spl-1 mutant allele11.

As previously described11, at ovule stage 2-I16, SPL/NZZ-GFP accumulated in the most apical cells of the L1 layer (Supplementary Fig. 1A). From stages 2-II to stage 2-V, associated with meiosis end, the SPL/NZZ-GFP accumulation domain comprised the L1 layer of the whole nucellus (Supplementary Fig. 1B–D). At later stages, the SPL/NZZ-GFP signal decreased without changing its pattern (Supplementary Fig. 1E, F).

We introduced the pSPL/NZZ::SPL/NZZ:GFP construct into the pAP1::AP1:GR ap1cal system for synchronised induction of flower development17,18. We performed DEX treatment on the inflorescences and evaluated the SPL/NZZ-GFP expression at different days after induction (DAI), to select samples prior to and at the MMC stage for collection. At 3 DAI, SPL/NZZ-GFP was mainly visible in another primordia, a result in line with SPL/NZZ activity in anther morphogenesis2,19 (Supplementary Fig. 1G–I). At 9 DAI, SPL/NZZ-GFP expression in anthers was almost absent (Supplementary Fig. 1J). By contrast, despite the partial loss of synchronisation at this later time point after AP1-GR induction, most flowers showed ovules at stage 2-I, where we could clearly observe a strong and specific nucellar SPL/NZZ-GFP accumulation (Supplementary Fig. 1K, L).

Therefore, we used three independent biological replicates of inflorescences at 9 DAI for Co-IP/MS, employing an anti-GFP antibody20. We found 660 proteins that were statistically enriched (log intensity difference >0; FDR≤0.05) in the IP samples with respect to the input controls (Supplementary Data 1). As expected, SPL/NZZ itself was found as one of the most enriched proteins (Fig. 1A, and Supplementary Data 1).

To obtain an overview regarding the function of the proteins enriched in the SPL/NZZ Co-IP/MS, we performed Molecular Function and Biological Process GO terms enrichment analysis. The most enriched categories were associated with RNA polymerase activity, DNA binding and transcription (Supplementary Data 1). Moreover, we found enrichment in Biological Process categories such as floral and gynoecium development, regulation of transcription and RNA processing (Supplementary Fig. 2A, and Supplementary Data 1).

Since SPL/NZZ binds to the DNA by its association with transcriptional factor (TF) complexes4,5, we focused our attention on the TFs identified among the SPL/NZZ partners. We found 56 TFs as putative SPL/NZZ interactors. Quite strikingly, among the TCPs, only TCP22 was statistically enriched in our list, while none of the YABBY TFs were present (Supplementary Data 1). The MADS-domain TF family was the most represented in our dataset, corresponding to almost 20% of all the TFs found (Fig. 1A, and Supplementary Data 1). This list of potential MADS-domain TFs interacting with SPL/NZZ included SEEDSTICK (STK), SHATTERPROOF2 (SHP2), SEPALLATA1 (SEP1), SEPALLATA2 (SEP2), SEPALLATA3 (SEP3) and AGAMOUS (AG), all proteins previously associated with ovule specification and development21–23.

Based on these results, we verified whether SPL/NZZ could directly interact with MADS-domain TFs in yeast. Since SPL/NZZ contains an EAR-motif, which could negatively impact GAL4 transcriptional activation functionality, we used an SPL/NZZ variant lacking the EAR-motif sequence (SPL/NZZΔ). We initially tested the interaction between SPL/NZZΔ, fused with a GAL4 activation domain (AD) or DNA binding domain (BD) with AG, SEP3Δ23 or STK. We found positive results for the SPL/NZZΔ - AG, SPL/NZZΔ - STK and SEP3Δ - SPL/NZZΔ interactions (Fig. 1B, and Supplementary Fig. 2B). It was previously shown that MADS-domain proteins generate multimeric complexes to regulate target genes22,24. Based on this, we performed yeast-three-hybrid assays. We initially evaluated the formation of complexes using SPL/NZZΔ fused to the AD, SEP3Δ to the TFT and either STK or AG to the BD, finding positive results for the SPL/NZZΔ - SEP3Δ - STK and SPL/NZZΔ - SEP3Δ - AG interactions (Fig. 1C). It is worth noting that, even though SPL/NZZΔ did not directly interact with SEP3Δ (Supplementary Fig. 2B) and only modestly with STK (Fig. 1B), the presence of all three components enhanced yeast growth, suggesting the formation of a more stable complex (Fig. 1C). We then tested the same interactions by fusing SPL/NZZΔ with the GAL4 BD, reconfirming the establishment of complexes with either STK and SEP3Δ or AG and SEP3Δ (Supplementary Fig. 2C). Concerning the screening with SPL/NZZΔ fused with the TFT, we found that the presence of SPL/NZZΔ did not enhance the already described SEP3-AG interaction22 (Supplementary Fig. 2D).

As we confirmed that SPL/NZZ can interact with the ovule identity MADS-domain TFs, we analysed STK, AG and SEP3 expression in the nucellus at stages 2-I, 2-II, concurrently with the MMC differentiation. STK and SEP3 were expressed in the nucellus, as shown by the study of GFP-fusion-protein reporter lines, with a pattern comparable to that of SPL/NZZ25–27 (Supplementary Fig. 2E–H). By contrast, AG was not expressed in the nucellus (Supplementary Fig. 2I, J)27. To further corroborate the SPL/NZZ direct interaction with SEP3 and STK, we performed Förster Resonance Energy Transfer-fluorescence lifetime imaging (FRET-FLIM)28 assay in planta. We transiently expressed, in tobacco leaves, SEP3Δ-GFP or STK-GFP fusion proteins either alone or in association with SPL/NZZΔ-mCHERRY (Fig. 1D–I, and Supplementary Fig. 3A–E). By imaging nuclei expressing solely SEP3Δ-GFP (Fig. 1D) or STK-GFP (Fig. 1G), we could measure, through FLIM, a GFP lifetime of around 2.3 and 2.7 ns, respectively (Fig. 1I, and Supplementary Fig. 3A, D). When SEP3Δ-GFP or STK-GFP were co-expressed with SPL/NZZΔ-mCHERRY (Fig. 1E, H), we measured a statistically significant reduction in the GFP lifetime because of FRET occurring between GFP and mCHERRY (Fig. 1I, and Supplementary Fig. 3B, E). Since FRET could occur only when donor and acceptor proteins are at a distance lower than 10 nm, this experiment confirms SEP3 and STK ability to physically interact with SPL/NZZ in plant cells.

It was already reported that STK-SEP3 can form a tetramer29 while SPL/NZZ can form a homodimer4. Using AlphaFold330, we predicted in silico the possible formation of SPL/NZZ-SEPs-STK complexes containing, respectively, two copies of SEP (1, 2, or 3), STK and SPL/NZZ. AlphaFold3 preliminary predictions could fold SPL/NZZ only from amino acid 61 to amino acids 91, forming an alpha helix, a protein portion that contains the 8 amino acids corresponding to the SPL-motif. Indeed, the SPL-motif was originally predicted to be part of an alpha helix spanning from amino acids 64 to amino acid 80, which has some homology to the first helix of MADS-domain TFs2. For these reasons, we considered only the SPL/NZZ region spanning from amino acid 61 to 91 for further predictions. All the different simulations predicted the formation of complexes with good confidence levels, assessing the reliability of the results obtained (Supplementary Fig. 4A). Regarding the docking features of SPL/NZZ to the MADS-domain TFs tetramer, the different simulations indicated that SPL/NZZ contacts the K-box of STK and SEPs (Supplementary Fig. 4B). Finally, we performed binding affinity analyses31–33, retrieving Gibbs free energy (ΔG) values and dissociation constants (Kd) that confirm the thermodynamic stability of the SPL/NZZ-MADS complexes (Supplementary Fig. 4C).

SPL/NZZ and the MADS-domain TFs share common targets

To identify the direct targets of SPL/NZZ, we used the pSPL/NZZ::SPL/NZZ:GFP AP1:GR ap1cal line to perform a ChIP-seq experiment. Three biological replicates of inflorescence at 9 DAI were used for chromatin immunoprecipitation using an anti-GFP antibody34,35. After input and IP samples sequencing, peaks were considered significant with a fold enrichment (FE) > 0, FDR≤0.05 and if present in at least 2 out of 3 biological replicates. This approach retrieved a list of 203 peaks, corresponding to 197 different genes (Supplementary Data 2). Around 80% of the peaks mapped in genomic regulatory regions, such as promoters, supporting the reliability of the results obtained (Fig. 2A). Among SPL/NZZ-bound genes, we found genes previously linked to SPL/NZZ function as ANT12, YUCCA6 (YUC6)36 and PHB37 (Supplementary Data 2). Interestingly, also SPL/NZZ itself was present in the list, indicating the possible existence of a feedback loop involved in SPL/NZZ regulation.

SPL/NZZ does not have a DNA-binding domain. This implies that SPL/NZZ binds to its direct targets via TF complexes in which it is recruited. To characterise which TFs mostly relate to SPL/NZZ activity, we performed a TF DNA-motif enrichment analysis. We used the MEME suite38 to analyse the 203 peak sequences retrieved from the SPL/NZZ ChIP-seq experiment. The only statistically enriched TF DNA binding site identified was the CArG-box consensus sequence, to which the MADS-domain TFs bind (Fig. 2B)39. In addition, enrichment analysis against the Transcriptional Factor Target lists from both the AGRIS server40–42 and the PlantGSAD database43 revealed that SPL/NZZ targets were enriched in genes targeted by MADS-domain TFs such as AG and SEP3 or floral homeotic factors such as APETALA2 (AP2) (Supplementary Data 2).

If SPL/NZZ binding to the DNA largely depends on its association with MADS-domain TF complexes, not only SPL/NZZ and MADS-domain TFs should target the same genes, but they should bind these genes in the same genomic sequences. Based on this assumption, we determined the overlap of peaks obtained by the SPL/NZZ ChIP-seq with the ones obtained from a SEP3 ChIP-seq dataset44. The already available SEP3 ChIP-seq was performed using the same induction system and tissue used for the SPL/NZZ ChIP-seq and on a similar stage of development (8 DAI). Over 90% of SPL/NZZ peaks (185 out of 203, corresponding to 182 different genes) overlapped with peaks in the SEP3 dataset, strongly supporting the cooperation between SPL/NZZ and SEP3 for binding to the DNA, at least at the developmental stages used for the ChIP-seq experiments. (Fig. 2C, D, and Supplementary Data 2).

Our data indicate that SPL/NZZ could cooperate with the MADS-domain TFs SEP1, 2, and 3, as well as STK and SHPs for MMC development. This outcome was also predictable, as it has been shown that MADS-domain TFs of classes C, D and E form multimeric complexes to establish ovule domains and define cell identity23. Regrettably, these transcription factors exhibit significant redundancy and multiple mutants, as the shp1 shp2 stk/+ one, displays ovules homeotically changed into leaves21–23, whereas the sep1 sep2 sep3 mutant has all floral organs homeotically changed into sepals45. Therefore, to investigate the potential relevance of these MADS-domain TFs in MMC differentiation, we created a pSPL/NZZ::MADSas construct to operate the post-transcriptional gene silencing of SEP genes in the nucellus (Supplementary Fig. 5A). We subsequently introduced this construct into shp1/shp1 shp2/shp2 stk/STK plants, intending to inhibit the activity of many ovule-identity MADS-domain TFs within the nucellus. We obtained three independent transformant lines expressing the pSPL/NZZ::MADSas (Supplementary Fig. 5B). Specifically, lines 1 and 2 resulted shp1 shp2 STK/STK, whereas line 3 was classified as a triple shp1 shp2 stk mutant. Although the phenotype was much less penetrant with respect to that reported in spl-1, the transgenic plants shp1 shp2 stk showed a ratio of 25% to 45% of ovules that failed to produce the MMC (Fig. 2E, F, and Supplementary Fig. 5C).

Altogether, these data support that SPL/NZZ and MADS-domain TFs cooperate, forming a multimeric complex for the differentiation of the MMC.

SPL/NZZ regulates the auxin signalling network

Having identified potential direct SPL/NZZ-SEP3 target genes, we focused on their biological function to determine the mechanisms involved in MMC differentiation. To do so, we performed a Biological Process GO terms enrichment analysis using the SPL/NZZ-SEP3 common targets (Supplementary Fig. 6A, and Supplementary Data 2). Besides categories associated with reproductive organs differentiation, the most enriched terms were related to auxin metabolism and biosynthesis, suggesting a direct link between SPL/NZZ and auxin (Supplementary Fig. 6A, and Supplementary Data 2).

To identify the genes whose expression was modified in the absence of SPL/NZZ, and the pathways associated with SPL/NZZ activity, we performed RNA-seq on wild-type (Col-0) and spl-1 mutant pistils at stage 946,47. Differential expression analysis retrieved a list of 6877 differentially expressed genes (DEGs) (FDR≤0.05) (Supplementary Data 3).

We performed a Biological Process GO terms enrichment analysis on the 3502 downregulated genes, revealing enrichment in terms associated with chromatin remodelling and floral development (Supplementary Fig. 6B, and Supplementary Data 3). By contrast, Biological Process GO terms enrichment analysis done on the 3375 upregulated genes confirmed an impairment in auxin biosynthesis, metabolism and signalling (Supplementary Fig. 6C, and Supplementary Data 3). We also observed enrichment in categories involved in cell cycle regulation, cell wall modification and callose deposition, processes known to be required for the MMC differentiation48. To further dissect the activity of SPL/NZZ over its direct targets, we analysed the SPL/NZZ-SEP3 common target list in light of the DEGs obtained from the spl-1 RNA-seq. We found that 77 SPL/NZZ-SEP3 targets were also differentially expressed in the spl-1 pistil with respect to the wild type (Supplementary Data 3). Nicely, these include genes involved in ovule development, such as ANT49, LUG50, AP251, CUC352, IPT114, SEP322, AGL1553 and FUL54. Interestingly, all the auxin biosynthetic genes present in the spl-1 RNA-seq, including also the genes targeted directly by SPL/NZZ, were upregulated (Supplementary Data 3). Key players of auxin biosynthesis, such as YUCCA2, 4, 6 and TAA1, which are normally expressed in the ovule chalaza55, showed enhanced expression. This result is in line with SPL/NZZ proposed activity as a transcriptional repressor, which is also supported by the reduced expression of YUC2 and 4 in the spl-D dominant mutant36. After biosynthesis in the chalaza, auxin is polarly transported toward the primordium apex mainly by PIN1, generating a region of high signalling output at the nucellus tip55–57. Auxin is then directed toward the L2 layer by PIN3 and PIN1 action1,57. Since it was already reported that PIN1 was drastically downregulated in spl-1 ovules14, as well as the auxin response15, we decided to investigate in more detail auxin transport and response in wild-type and spl-1 ovules to evaluate their impact on the spl-1 phenotype.

Focusing on wild-type ovules, we observed that the PIN1-GFP signal increased in the nucellus from stage 2-I, the stage in which the differentiation of the MMC initiates, to stage 2-III, associated with the complete differentiation of the MMC (Fig. 3A, B, M). Likewise, we measured a significant increase in the nucellar DR5v258 signal passing from stage 2-I to stage 2-III (Fig. 3E, F, N). This dynamic in PIN1 and DR5v2 expression during MMC differentiation could suggest the requirement of auxin transport and signalling to support the MMC identity acquisition. Indeed, even though we observed that PIN1-GFP accumulated similarly in wild-type and spl-1 ovules at stage 2–I (Fig. 3A, C, M), at stage 2-III, PIN1-GFP signal was drastically reduced in the spl-1 mutant (Fig. 3B, D, M), as previously shown14,15. Likewise, the DR5v2 signal observed between wild-type ovules at stages 2-I and 2-III was severely reduced in the spl-1 background (Fig. 3E–H, N). Besides PIN1, PIN3 is also involved in auxin transport in the nucellus. Despite this, the pPIN3::PIN3:GFP57,59 signal was localised at the very tip of ovule primordia in spl-1, and no differences were observed compared to the wild type (Supplementary Fig. 7A–D).

Downregulation of PIN1 expression and DR5v2 response suggests that auxin is accumulated at lower levels in the spl-1 nucellus. Indeed, by using the R2D2 reporter line58, we detected a higher accumulation of DII-VENUS in the spl-1 nucellus with respect to the wild type (Fig. 3I–L, and Supplementary Fig. 7E–I).

Considering the behaviour of PIN1 and DR5v2, together with the fact that auxin transport was previously suggested to be required for MMC development15, we believed that PIN1 downregulation could be the main cause of MMC absence in the spl-1 mutant ovule.

Restoring the auxin transport is sufficient to rescue the MMC differentiation in spl-1 ovules

Even if molecularly SPL/NZZ is a repressor of transcription, during MMC differentiation, SPL/NZZ works as a positive modulator of PIN1 expression. This implies that SPL/NZZ activity over PIN1 is indirect and involves, potentially, the direct targeting of negative regulators of PIN1. Indeed, PIN1 was not present in the list of SPL/NZZ direct targets. Taking into consideration this information, among SPL/NZZ-SEP3 direct targets, we searched for factors which could be genetically associated with SPL/NZZ activity, and which could work as intermediates of SPL/NZZ action over PIN1. During ovule patterning, SPL/NZZ was shown to be required for the repression of chalazal factors such as ANT, BEL1, INO and PHB in the nucellus12,37,49. As mentioned above, ANT is a direct target of the SPL/NZZ-MADS-domain TF complex. Particularly, in both SPL/NZZ and SEP3 ChIP-seq datasets, we found a peak containing 4 CArG-box sequences at around -3.6 Kb from ANT TSS (Supplementary Fig. 8A). Absence of SPL/NZZ function determines ANT ectopic expression in the nucellus12 (Supplementary Fig. 8B–I). Our attention was caught by ANT as a putative intermediary of SPL/NZZ action over PIN1, especially due to the previous description of the ability of ant mutation to rescue MC differentiation in spl/nzz mutant background12. Considering the centrality of auxin transport and response for the MMC differentiation, we speculated that ANT could act as a repressor of PIN1, contributing to PIN1 downregulation in the spl-1 nucellus. Even though we did not find PIN1 among ANT direct targets60, we observed that PIN1 was ectopically expressed in the chalaza and funiculus of ant.4 mutant ovules with respect to the wild type (Supplementary Fig. 8J–Q). This suggests that, in ovules, ANT acts as an indirect local repressor of PIN1.

Based on our present and previously reported observations15, we hypothesised that restoring polar auxin transport in the spl-1 ovule might be sufficient to recover the MMC differentiation. Therefore, we developed the pSPL/NZZ::PIN1 construct, and we transformed it into spl-1 mutant plants. Four distinct spl-1 lines expressing the pSPL/NZZ::PIN1 construct were characterised (Supplementary Fig. 9A).

In contrast with the spl-1 situation (Fig. 4F–J, P), in spl-1 pSPL/NZZ::PIN1, ovule developmental defects were almost completely restored (Fig. 4A–E, K–P, and Supplementary Fig. 9B). The MMC correctly developed (Fig. 4C, D, M, N) and completed the megasporogenesis (Fig. 4E, O). Indeed, fertile ovules were formed and fertilised by pollen produced by the same plants, showing that the pSPL/NZZ:PIN1 had also rescued the PMC defects (Supplementary Fig. 9C). Interestingly, spl-1 pSPL/NZZ:PIN1 ovules also showed a rescue of spl-1 integuments defect (Fig. 4G–J, L–O). Indeed, ovules could correctly differentiate inner and outer integuments (Fig. 4G–J, L–O). Complementation of inner integuments development suggests that SPL/NZZ is important for the proper PIN1 localisation and auxin transport during inner integuments formation. Indeed, regulation of auxin transport was shown to be involved in integuments development61. Nevertheless, similarly to the spl-1 mutant, the funiculus length in spl-1 pSPL/NZZ::PIN1 ovules remained statistically longer with respect to the wild type (Supplementary Fig. 10A, B). We hypothesised that this phenotype could be related to the fact that genes involved in funiculus differentiation remained deregulated in spl-1 pSPL/NZZ::PIN1 ovules. For instance, it was shown that ANT ectopic expression is directly associated with the differentiation of a longer funiculus62. By ISH, we observed that ANT remained ectopically expressed in both nucellus and funiculus of spl-1 pSPL/NZZ::PIN1 ovules (Supplementary Fig. 10C). The ectopic expression of ANT in spl-1 pSPL/NZZ::PIN1 not only provides a hierarchical view in which PIN1 is placed downstream ANT but also represents a plausible explanation for the elongated funiculus of spl-1 pSPL/NZZ::PIN1 ovules.

The inhibition of ARFs activity phenocopies the spl-1 mutation

The results described above demonstrate the need for SPL/NZZ to regulate the auxin accumulation in the nucellus, which is essential for MMC differentiation. According to the auxin response model, auxin accumulation enhances Aux/IAAs degradation, releasing the ARFs from Aux/IAAs repressing activity63. This leads to the regulation of the ARFs target genes expression. Based on this model, we hypothesised that the impairment of the MMC differentiation in the spl-1 mutant could be ascribed to the post-translational suppression of ARFs function. Indeed, lower auxin accumulation in spl-1 may inhibit Aux/IAAs degradation, affecting ARFs activity and, thus, MMC differentiation. Accordingly, ovules containing an Aux/IAA isoform resistant to the auxin-dependent proteasomal degradation might mimic the spl-1 phenotype. Single-cell RNA-seq, performed using ovules between stages 2-I and 2-III, revealed that Aux/IAA1, 2, 3, 4, 8, 9, 13, 16 and 26 were expressed in nucellar clusters64. To select the appropriate Aux/IAA to repress the ARFs activity in the nucellus, we chose the one that was previously reported to interact with several nucellar-expressed ARFs65–67, and for which a dominant auxin-resistant allele was available. We chose the Aux/IAA3/SHY268 dominant mutant allele shy2.669. shy2.6 carries a mutation in the degron motif, impairing its ability to interact with the SCF/TIR1 complex at threshold auxin levels69, remaining constitutively bound to ARFs PB1 domain69. Consequently, we generated the pSPL/NZZ::shy2.6 construct to express shy2.6 in the nucellus of wild-type ovules.

We introduced the pSPL/NZZ::shy2.6 construct into wild-type plants, obtaining three independent lines expressing the transgene (Supplementary Fig. 11A). Even if with a lower penetrance with respect to the spl-1 mutant, in all the lines we observed an impairment in MMC differentiation (Fig. 5A–M). To confirm that the observed phenotype was caused by the inhibition of ARFs activity rather than other factors, such as a disruption of the polar auxin transport, we examined the expression of pPIN1::PIN1:GFP in pSPL/NZZ::shy2.6 plants. The expression of PIN1-GFP in pSPL/NZZ:shy2.6 resembled that of the wild type, suggesting that the observed phenotype was probably caused by an impairment in ARFs activity (Supplementary Fig. 11B–E).

ARFs activity is required for the MMC differentiation

Collectively, our data support a scenario in which the MMC differentiation is mediated by the post-translational regulation of the ARFs action. SHY2 can interact with all the class A ARFs, including ARF5/MP, ARF6, ARF7, ARF8 and ARF19. Concerning the repressor ARFs, SHY2 was shown to interact with ARF4 and ARF965–67. According to an ovule single-cell RNA-seq dataset64, MP, ARF6, ARF8 and ARF9 are the only SHY2-interacting ARFs expressed in the nucellus. Consequently, we analysed MP, ARF6, ARF8 and ARF9 expression in both wild-type and the spl-1 ovules. We used previously published reporter lines to study MP, ARF6 and ARF8 domains of transcription and protein accumulation70. Concerning ARF9, as no functional protein reporter lines were available, we studied its expression pattern by in-situ hybridisation.

In ovule primordia, the levels of both ARF6-VENUS and ARF8-VENUS were presumably controlled at the post-translational stage, supporting previously reported observations53 (Supplementary Fig. 12E–L). Indeed, although both genes were transcribed in ovules64 (Supplementary Fig. 12E, I, G, K), ARF6 was faintly visible solely in a limited number of cells (Supplementary Fig. 12F, H), while ARF8 was not visible (Supplementary Fig. 12J, L). By contrast, the MP-VENUS protein accumulated in the nucellus, as previously shown70,71 (Supplementary Fig. 12A–D). The three Class A ARFs exhibited comparable expression and protein accumulation in both wild-type and spl-1 ovules (Supplementary Fig. 12A–L). Concerning ARF9, we detected its transcript accumulation in both wild type and spl-1 nucellus at stage 1-II (Supplementary Fig. 13A, C). At stage 2-III, the ARF9 transcript was accumulated in the MMC and the integuments of wild-type ovules (Supplementary Fig. 13B). By contrast, in the spl-1 mutant background, ARF9 transcript appeared miss-localised in both the nucellus and the chalaza (Supplementary Fig. 13D). This could be mainly due to the fact that, in a wild-type ovule, ARF9 transcript accumulates in the MMC and the integuments, both structures that are impaired in the spl-1 background. Despite this, by analysing the spl-1 RNA-seq, we observed an overall decrease in ARF9 expression level in the spl-1 pistil (Supplementary Data 3).

Given their expression patterns, MP and ARF9 were selected as ARFs potentially involved in the downstream auxin response, required for MMC differentiation. According to our hypothesis, the absence of the MMC in the spl-1 mutant was associated with a decrease in the nucellar auxin response, caused by the stabilisation of Aux/IAAs and the consequential ARFs inhibition. Indeed, as previously described63,70,72,73, ARFs activity is post-translationally regulated by the balance between ARFs and Aux/IAAs relative abundance. For this reason, we hypothesised that increasing MP or ARF9 levels in the spl-1 background could overcome the repression operated by the Aux/IAAs, restoring the MMC presence. Therefore, we generated the pSPL/NZZ::MP and the pSPL/NZZ::ARF9 constructs that we introduced in the spl-1 mutant. We obtained two independent spl-1 pSPL/NZZ::MP lines, while we found three spl-1 pSPL/NZZ::ARF9 independent lines (Supplementary Fig. 14A, B). In spl-1 pSPL/NZZ::MP plants, we observed that ~10% of ovules developed an MMC (Fig. 6A–F, I). However, these cells did not undergo meiosis, suggesting only a partial acquisition of the MMC identity (Supplementary Fig. 14C). By contrast, the majority of spl-1 pSPL/NZZ::ARF9 ovules differentiated an MMC, which could also proceed into sporogenesis (Fig. 6G, H, J, and Supplementary Fig. 14D), indicating the direct involvement of ARF9 in the MMC differentiation.

Altogether, our results highlight the regulation of the ARFs action as a key step of the SPL/NZZ downstream response during MMC differentiation.

Methods
Plant material and growing conditions
A. thaliana Columbia-0 plants were grown in controlled conditions under a long-day photoperiod (16 h light 150 µm.m−2.s−1 / 8 h dark) at 22°C. spl-1 mutant in the Col-0 background was previously described14. The pSPL/NZZ::SPL/NZZ:GFP line was previously described11. The AP1:GR/ap1cal inducible system was described in ref. 17. shp1, shp2, stk/STK plants were described in ref. 21. ant.4 mutant in the Ler background was previously described78,79. pSTK:STK:GFP, pSEP3::SEP3:GFP, pAG::AG:GFP were previously described25–27. The spl-1 pPIN1::PIN1:GFP line was described in ref. 14. pPIN3::PIN3:GFP59, DR5v258 and R2D258 lines were manually crossed with spl-1 heterozygous plants.

Generation of transgenic lines
To obtain the pSPL/NZZ::SPL/NZZ:GFP pAP1::AP1:GR ap1cal inducible line, pAP1::AP1:GR ap1cal plants17 were transformed with the pSPL/NZZ::SPL/NZZ:GFP construct via the floral dip technique using the Agrobacterium tumefaciens strain GV3101.

SPL/NZZ:GFP AP1:GR ap1cal plants were induced with dexamethasone treatment directly on the inflorescence-like meristem for 9 days. After the third and the ninth dexamethasone treatment, the expression of SPL/NZZ-GFP was controlled with a confocal microscope.

The pSPL/NZZ::MADSas, pSPL/NZZ::PIN1, pSPL/NZZ::shy2.6, pSPL/NZZ::MP, pSPL/NZZ::ARF9 and pSPL/NZZ::ANT constructs were obtained with the multisite gateway cloning technique. MADSas, PIN1, shy2.6, MP, ARF9 and ANT CDS sequences were amplified using the Q5 High-Fidelity DNA polymerase (NEB) and cloned into the pDONR221 plasmid, according to the BP II protocol (Invitrogen, cat. number: 11789020). Multi-site LR reactions were performed using the pDONR221 P1-P4 containing the SPL/NZZ promoter sequence11; the pDONR221 containing either MADSas, PIN1, shy2.6, MP, ARF9 or ANT; the pDONR221 P3-P2 carrying the SPL/NZZ 3’UTR sequence11 and the destination vector pH7m34GW80, according to the LR II Plus protocol (Invitrogen, cat. number: 12538120). Thanks to the floral dip technique using the Agrobacterium tumefaciens strain GV3101, pSPL/NZZ::PIN1, pSPL/NZZ::MP and pSPL/NZZ::ARF9 were transformed into the SPL/NZZ spl-1 heterozygous background. Four, two and three T2 independent transformant lines were analysed for each transformation, respectively. pSPL/NZZ::MADSas was transformed into the shp1, shp2, stk/STK background. Three T1 independent transformant lines were analysed.

pSPL/NZZ::shy2.6 and pSPL/NZZ::ANT were transformed into wild-type Col-0 plants. Three T1 independent transformant lines were analysed for each transformation. Primers used are listed in Supplementary Data 4.

RT-qPCR expression analysis
For each sample, 5 inflorescences pooled together (comprising flowers spanning from stage 1 to stage 12) were used for total RNA extraction. RNA extraction was performed using the Macherey-Nagel NucleoSpin RNA plus kit (Item number: 740984.50) according to the manual instructions. 500 ng of DNA-clean total RNA for each sample was retrotranscribed using the Bio-Rad iScript™ Reverse Transcription Supermix for RT-qPCR (Bio-Rad, cat number: 1708840). cDNA was used for RT-qPCR using the Bio-Rad iTaq Universal SYBR Green Supermix (Bio-Rad, cat number: 1725120). ACTIN8 was used as a housekeeping normaliser for relative normalised expression calculation. Expression levels were expressed as mean ± SEM among three technical replicates. Primers used are listed in Supplementary Data 4.

Analysis of ovule morphology by confocal and optical microscopy
Ovule morphology was evaluated by both confocal and optical wide-field microscopy. For confocal microscopy, pistils were manually dissected from the flowers and mounted in a solution of 10 µg/ml Renaissance 2200 (Renaissance Chemicals, SR2200). Samples were immediately imaged using a Nikon AX confocal microscope. Renaissance 2200 was excited at 405 nm, and the emission signal was detected between 440 and 470 nm. For wide field microscopy, inflorescences were collected in a solution of 9:1 ethanol/acetic acid. After 24 h at 4°C, inflorescences were transferred to a solution of 70% ethanol. Pistils were manually dissected and mounted in chloral hydrate clearing solution before microscopic imaging. Analysis of the percentage of ovules developing the MMC in wild type, spl-1, pSPL/NZZ::MADSas, spl-1 pSPL/NZZ::PIN1, pSPL/NZZ::shy2.6, spl-1 pSPL/NZZ::MP, spl-1 pSPL/NZZ::ARF9 and pSPL/NZZ::ANT was performed on ovules at stages 2-II, 2-III.

For fluorescent reporter lines signal imaging, pistils were mounted in the Renaissance 2200 solution and imaged using a Nikon AX confocal microscope. mTQ was excited with a 445 nm laser and detected at 480–490 nm. GFP was excited with a 488 nm laser and detected at 520-540 nm. VENUS was excited with a 514 nm laser and detected at 530–560 nm. tdTOMATO was excited with a 514 nm laser and detected at 570-630 nm. Imaging was performed with sequential settings to eliminate signal crosstalk. Confocal images were analysed with the Fiji software81. For PIN1-GFP and DR5v2 signal quantification, the Renaissance 2200 channel, showing the cellular organisation, has been used to define a region of interest (ROI) comprising the whole L1 layer of the nucellus. The mean GFP and tdTOMATO signals were subsequently measured in the ROI. The R2D2 signal was analysed as previously described70. The tdTOMATO channel has been used to generate a binary mask indicating the nucellar nucelli showing R2D2 expression. The VENUS and the tdTOMATO mean signals in the single nuclei were subsequently measured.

Co-IP/MS material generation and data analysis
IP-MS experiments were performed as described before20. Essentially, nuclei were isolated from the unfixed plant tissues. Proteins, including native protein complexes, were extracted from nuclear extracts and immunoprecipitated with the GFP antibody using µMACS GFP Isolation Kit (Miltenyi Biotec, cat. number: 130-091-288). Immunoprecipitated proteins were released from the beads with 8 M urea. As control, protein extracts from before the immunoprecipitation were used and processed with the FAST method82. Eluted proteins were reduced with DTT, alkylated with IAA and digested with Trypsin/Lys-C Mix, Mass Spec Grade (Promega, cat. number: V5071). Resulting peptides were desalted, dried and resuspended in a loading solution. Peptide concentration was estimated using the NanoDrop One A205 method. Samples were analysed using the EASY-nLC 1200 (Thermo Fisher ScientificTM) coupled to the Q Exactive Plus mass spectrometer (Thermo Fisher ScientificTM). Peptides were loaded onto the Acclaim PepMap 100 C18 analytical column and separated with a linear solvent B gradient of 5% to 40% over 70 min and 40% to 50% over 5 min. Tandem MS spectra (MS/MS) were acquired in the data-dependent acquisition (DDA) mode using a TOP15 approach.

The RAW peptide sequencing data obtained from the Q Exactive Plus were processed by the MaxQuant (version 1.6.14.0) software. Settings of the MaxQuant were mostly kept default with “label-free quantification (LFQ)”, “iBAQ” and “Match between runs” options on. The protein database was A. thaliana UniProt UP000006548 without protein isoforms. After the MaxQuant analysis, the output proteinGroups.txt file was taken for further filtering and statistical analysis in the Perseus (version 1.6.15.0) software. The protein LFQ intensity data were log2-transformed. Proteins identified by only modified peptides (“Only identified by site”), by reverse sequence database (“Reverse”) and protein contaminants (“Potential Contaminant”) were removed from the analysis. Proteins with LFQ intensity values in at least two biological replicates and proteins with at least two unique peptides were kept. Missing protein LFQ intensity values were replaced only in the input samples by the imputation from the left arm of the LFQ intensity normal distribution (0.3 width, 1.8 downshift, separately for each column) to emulate background protein level. Relative protein amount differences were evaluated by two-sample t-tests with permutation-based FDR correction and S0 parameter set to 1.0. The GO term enrichment analyses were performed with clusterProfiler83, with default parameters. The statistical significance of enriched categories was determined by the Benjamini–Hochberg (False Discovery Rate-FDR) test84, considering significant only categories with FDR≤0.05. GO term enrichment analyses were performed on R studio, version 4.4.1.

ChIP-seq material generation and data analysis
ChIP-seq experiments were performed as described before34,35. Specifically, plant tissues were fixed in 1% formaldehyde solution, frozen in liquid nitrogen and ground to a fine powder. Nuclei were isolated through a sequential wash with buffer containing 0.5% Triton X-100 and lysed followed by chromatin sheering by sonication. After chromatin clearing, immunoprecipitation was performed with the GFP antibody (Abcam, ab290) followed by incubation with protein A magnetic beads (Thermo Fisher ScientificTM, cat. number: 88845). The immunoprecipitated chromatin was released from the beads and de-crosslinked. The input sample, corresponding to the protein extract from before the immunoprecipitation, was also de-crosslinked and used as a ChIP control. The DNA was purified and concentrated using DNA Clean & Concentrator-5 kit (Zymo Research, cat. number: D4004) following manufacturer’s instructions and eluted with 20 µl of the DNA Elution Buffer. The DNA sequencing libraries for both ChIP and Input samples were prepared using ThruPLEX DNA-Seq Kit (Takara Bio, cat. number: R400674) and SMARTer DNA Unique Dual Index Kit (Takara Bio, cat. number: R400697) following manufacturer’s instructions. DNA double size selection 100–700 bp was performed using SPRI select beads (Beckman Coulter, cat. number: B23317). DNA libraries were sequenced on the Illumina sequencer.

Raw data were trimmed using Trimmomatic 0.3985 with the following parameters: ILLUMINACLIP:1:30:6, LEADING:3, TRAILING:3, HEADCROP:10, AVGQUAL:20, MINLEN:35. Mapping was performed using Bowtie 2 (version 2.5)86 on TAIR10 genome. Duplicated reads were removed using SAMtools (version 1.7)87. The peak calling was performed with MACS2 (version 2.2.9.1)88 with -g 118459858. Peaks enrichment statistical significance was evaluated according to the Benjamini–Hochberg (FDR) test. Peaks were merged using BEDTools (version 2.26.0)89. Only peaks present in at least 2 out of 3 biological replicates were considered. Peaks annotation was performed with ChIPseeker90 with the following parameters: tssRegion comprised between −3000 and 0 and the genomic Annotation Priority to 5 UTR, 3 UTR, Exon, Intron, Promoter, Downstream and Intergenic. The GO term enrichment analysis was performed with clusterProfiler83, with default parameters. The statistical significance of enriched categories was determined by the Benjamini–Hochberg (FDR) test, considering significant only categories with FDR≤0.05. Peak annotation and the GO term enrichment analysis were performed on R studio, version 4.4.1. The consensus sequence enrichment analysis was performed using the MEME suite38 with default parameters. The analysis of common peaks between the SPL/NZZ ChIP-seq and the SEP3 ChIP-seq has been performed by using the command subsetByOverlaps on R studio, version 4.4.1.

In-silico prediction of protein complex structures
Protein complex structure predictions were performed with AlphaFold3 algorithm with default parameters. Protein sequences and domains annotation of SPL/NZZ (AT4G27330), STK (AT4G09960), SEP1 (AT5G15800), SEP2 (AT3G02310) and SEP3 (AT1G24260) were retrieved from the Uniprot database. AlphaFold3 models were analysed with the PyMOL software. The complexes confidence-level representations are based on the confidence files provided by AlphaFold3 algorithm. The binding-affinity analysis and the visualisation of the SPL/NZZ binding interface with the MADS tetramer was performed with the Prodigy webserver31–33 using default parameters, at a temperature of 22°C.

RNA-seq material generation and data analysis
Three biological replicates of wild-type and spl-1 pistils at stage 947 were collected in the NucleoProtect RNA (Macherey-Nagel, cat. number: 740750.500) and stored at 4°C for 1 day. RNA extraction was performed using the NucleoSpin RNA kit (Macherey-Nagel). RNA was sequenced with the Novaseq X, at a depth of 30 M reads with a paired-end sequencing.

Raw data were trimmed using Trimmomatic 0.3985 with the following parameters: ILLUMINACLIP:1:30:6, LEADING:3, TRAILING:3, HEADCROP:10, AVGQUAL:20, MINLEN:35. Mapping was performed with STAR91, with --alignIntronMax 10000 on TAIR10 genome. Counts were obtained with featureCounts92 (version 2.0.1), using Araport11 annotation.

Counts were filtered with HTSFilter93 and the DEGs were obtained using DESeq294, considering statistically differentially expressed DEGs with an FDR≤0.05, as determined by the Benjamini–Hochberg (FDR) test. The GO terms enrichment analyses were performed with clusterProfiler83. Only categories with an FDR≤0.05, according to the Benjamini–Hochberg (FDR) test were considered statistically significant. DEGs evaluation and GO terms enrichment analyses were performed on R studio, version 4.4.1.

In-situ hybridisation
In-situ hybridisation was performed as described in refs. 54,95, with some modification. Briefly, developing inflorescences were fixed in Formaldehyde/Ethanol/Acetic Acid (3.7%/50%/5%) solution, dehydrated and embedded in paraffin. Embedded inflorescences were sectioned to 8 µm thickness with a microtome. Dewaxing in histoclear was followed by rehydration and acidification in 0.2 M HCl for 20 min. Neutralisation occurs in 2X SSC for 10 min, followed by an incubation for 30 min with 1 µg/ml Proteinase K at 37°C. Glycine (2 mg/ml) was used to incubate sections for 5 min to block Proteinase K, followed by 10 min of post-fixation in 4% formaldehyde. Washing in PBS and dehydrating via an ethanol series occurs before the application of the hybridisation solution (100 µg/ml tRNA; 6X SSC; 3% SDS; 50% formamide, containing ~100 ng/µl of antisense DIG-labelled RNA probe). Samples were hybridised overnight at 48°C and the next day washed in SSC 2X/formamide 50% solution for 2 times for 1 h each. Antibody incubation (Anti-Digoxigenin-AP, Fab fragments, Roche, cat. number: 11093274910) and colour detection (BCIP/NBT Colour Development Substrate, Promega, cat. number: S3771) were performed according to the manufacturer’s instructions. ANT, PIN1 and ARF9 anti-sense probes were obtained from retrotranscribed RNA extracted from wild-type inflorescences. Primers used are listed in Supplementary Data 4.

Yeast-two- and three-hybrid assays
Yeast-two- and three-hybrid assays were performed following the protocol from ref. 96 with some modifications.

Both yeast-two- and three-hybrid assays were performed at 28°C using a single AH109 yeast strain via co-transformation, or AH109 and Y187 yeast strains through co-transformation and mating.

The interactions were tested in selective media lacking leucine, tryptophan, histidine and adenine. AG and STK full-length CDS and SPL/NZZ and SEP3Δ shorter versions (SPL/NZZΔ and SEP3Δ, respectively) were cloned into Gateway vector GAL4 system (pGBKT7 and pGADT7, Clontech; pTFT197). Primers used are described in Supplementary Data 4.

FRET-FLIM
To test SPL/NZZ, SEP3 and STK interaction by FRET-FLIM, SPL/NZZΔ, SEP3Δ and STK CDS sequences, without the stop codons, were cloned into the pDONR207 plasmid according to the BP II protocol (Invitrogen, cat. number: 11789020). Thanks to LR II Plus reaction (Invitrogen, cat. number: 12538120), SEP3Δ and STK CDS were introduced into the pAB117 (provided by Rüdiger Simon, HHU Düsseldorf), which allow the C-terminus fusion of the GFP. SPL/NZZΔ was introduced in the pAB118 (provided by Rüdiger Simon, HHU Düsseldorf), which allows the C-terminus fusion of the mCHERRY. SEP3Δ was also cloned into the pAB119 plasmid (provided by Rüdiger Simon, HHU Düsseldorf) which allow the C-terminus fusion with both GFP and mCHERRY. Plasmids were transformed in the Agrobacterium tumefaciens strain GV3101. Tobacco leaf infiltration was performed as previously described98. The effect of FRET between GFP and mCHERRY was measured by FLIM of the GFP, using a Nikon ECLIPSE Ni-E A1 confocal equipped with a pulsed laser at 485 nm and taking advantage of the SymPhoTime 64 software (PicoQuant, https://www.picoquant.com/). Each acquisition has been performed reaching a photon count per pixel spanning from 30 to 50. Acquisition of coumarin 6 (0.1 mM EtOH 100%), was used as a reference. Coumarin 6 has excitation and emission spectra similar to the GFP ones and has a known photon lifetime of around 2.5 ns. The evaluation of its decay was used, through a phasor transformation function, to calculate the Instrument Response. The FLIM results obtained by the SymPhoTime 64 software were corrected with the Instrument Response thanks to the FLIM-Phasor analysis software99 (FlimLabs, https://www.flimlabs.com), generating a phasor plot in which the different pixels of an image are clustered in relation to their lifetime. This step allow the identification of the nuclei and the evaluation of the GFP photon decay.

Statistical information
The SPL/NZZ Co-IP/MS experiment has been performed on three independent biological replicates of pSPL/NZZ::SPL/NZZ:GFP pAP1::AP1:GR ap1cal inflorescences at 9 DAI. Proteins with LFQ intensity values in 2 out of 3 biological replicates and with at least two unique peptides were kept. Relative protein amount differences were evaluated by two-sample t-tests with permutation-based FDR correction and S0 parameter set to 1.0.

The SPL/NZZ ChIP-seq experiment has been performed on three independent biological replicates of pSPL/NZZ::SPL/NZZ:GFP pAP1::AP1:GR ap1cal inflorescences at 9 DAI. Peaks with an FDR≤0.05 and present in at least 2 out of 3 biological replicates were considered. Statistical significance was evaluated according to the Benjamini–Hochberg (FDR) test.

Statistical significance of SPL/NZZ target genes enrichment against AGRIS and PlantGSAD transcriptional factor target lists has been evaluated with the ShinyGo100 web tool, and only categories with an FDR≤0.05 were considered significant.

The spl-1 vs wild type RNA-seq experiment has been performed on three independent biological replicates of wild type and spl-1 pistils at stage 9 of development. Statistical significance of spl-1 vs wild-type DEGs was evaluated with the Benjamini–Hochberg (FDR) test and only genes with an FDR≤0.05 were considered significant.

Statistical significance of Gene Ontology enrichment analyses on the results obtained from the SPL/NZZ Co-IP/MS, the SPL/NZZ ChIP-seq and the spl-1 vs wild type RNA-seq, was evaluated according to the Benjamini–Hochberg (FDR) test. Categories with an FDR≤0.05 were considered significant.

In the FRET-FLIM experiment to test the SEP3-SPL/NZZ interaction, statistical significance of the reduction in the GFP lifetime has been evaluated according to the results of the student’s t-test, two-sided distribution, homoscedastic, comparing the GFP lifetime in nuclei expressing SEP3Δ-GFP with the one measured in nuclei expressing SEP3Δ-GFP+SPL/NZZΔ-mCHERRY. The comparison between SEP3Δ-GFP and SEP3Δ-GFP-mCHERRY has been used as a positive control for the experiment. Comparisons with a p≤0.05 were considered significant. student’s t-test calculation has been performed with Microsoft Excell. In the FRET-FLIM experiment to test the STK-SPL/NZZ interaction, statistical significance of the reduction in the GFP lifetime has been evaluated according to the results of the student’s t-test, two-sided distribution, homoscedastic, comparing the GFP lifetime in nuclei expressing STK-GFP with the one measured in nuclei expressing STK-GFP+SPL/NZZΔ-mCHERRY. The comparison between SEP3Δ-GFP and SEP3-GFP-mCHERRY has been used as a positive control for the experiment. Comparisons with a p≤0.05 were considered significant. student’s t-test calculation has been performed with Microsoft Excell. For PIN1-GFP, DR5v2, and R2D2 signal intensity measurements, statistical significance has been evaluated according to the results of the student’s t-test, two-sided distribution, homoscedastic, comparing the mutant with the wild-type. Comparisons with a p≤0.05 were considered significant. student’s t-test calculation has been performed with Microsoft Excell. For the morphological analyses of ovules differentiating an MMC, as well as funiculus length measurements, statistical significance has been evaluated according to the results of one-way ANOVA with post-hoc Tukey HSD test. Comparisons with a p≤0.05 were considered significant. The analysis has been performed with the web tool: https://astatsa.com/OneWay_Anova_with_TukeyHSD/. Exact p-values can be found in the Source Data.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript by Cavalleri et al. investigates the role of SPL/NZZ in regulating megaspore mother cell (MMC) development by identifying ovule-identity MADS-domain transcription factors as direct targets, co-regulating an auxin-dependent downstream network. SPL/NZZ is recognized as a master regulator in plant reproduction, particularly in the differentiation of MMCs and PMCs. In this manuscript, the authors performed Co-IP/MS of SPL/NZZ and identified several MADS-domain transcription factors as SPL/NZZ-specific interacting proteins during the early MMC stages of ovule development. The authors then verified their interactions in yeast, supporting the formation of multimeric protein complexes. ChIP-seq assays show that SPL/NZZ and the MADS-domain TFs share common targets, over 90%, indicating that they might function in the same pathway to regulate the MMC development. Through Biological Process GO term enrichment analysis done on these target genes, they made a direct link between SPL/NZZ and auxin. Furthermore, using loss-of-function mutants and complementation assays it is shown that the severe developmental defects observed in the MMC differentiation in spl-1 mutant ovules could be due to the impairments in PIN1-mediated auxin transport and accumulation, and Aux/IAA-ARF module-mediated auxin transcriptional responses. Together the multi-omics, genetic, and cell biological data conclusively demonstrate that SPL/NZZ acting with MADS-domain transcription factors determines the specification and development of the MMC via an auxin-dependent downstream network.\nIn general, the manuscript reads well and results are presented in appropriate figures. Several aspects warrant further clarification and additional experiments to strengthen the study’s claims.\n1) Line126: The authors used yeast-two- and three-hybrid assays to confirm the interactions between SPL/NZZ and MADS-domain TFs. To provide more robust evidence of these interactions in their native context, fluorescence resonance energy transfer (FRET) or fluorescence lifetime imaging microscopy (FLIM-FRET) analyses in living plant cells, especially during MMC differentiation, are recommended.\n2) Line 153: The manuscript references Alphafold predictions to suggest the formation of SPL/NZZ-SEPs-STK complexes. To substantiate these predictions, detailed docking site features and docking scores should be presented, offering insights into the potential structural configurations of these protein interactions.\n3) Line 189: With over 90% overlap between SPL/NZZ and SEP3 ChIP-seq peaks, qRT-PCR assays should be conducted on representative target genes, particularly those involved in the ovule specification and development, to validate the ChIP-seq findings.\n4) Line 219: Why did the authors choose to evaluate auxin transport, accumulation, and response concerning SPL/NZZ activity, but not auxin biosynthesis? The rationale for this is not fully logical, as some data suggest that SPL/NZZ function is closely linked to auxin biosynthesis. RNA-seq assay shows an impairment in auxin biosynthesis in the spl-1 mutant. SPL/NZZ bound genes include the gene YUCCA6, which might be a direct target of SPL/NZZ, as later data show that SPL/NZZ-ANT module regulation over PIN1 is indirect in ovule development.\n5) Line 227: The signal of PIN1-GFP in wild-type and spl-1 ovules should be quantified (Figure 3A-D).\n6) Line 233: Investigating whether mutants of MADS-domain TFs, such as sep mutants, exhibit impaired auxin response similar to the spl-1 mutant could reveal functional overlaps and distinctions within this regulatory network.\n7) Line 265: The complementation assays demonstrating PIN1’s ability to rescue ovule developmental defects in spl-1 mutants would benefit from the inclusion of negative controls, such as inactive versions of PIN1, to confirm the specificity of the observed rescue effects. Additionally, it would be valuable to assess whether the application of exogenous auxin can rescue the developmental defects observed in the spl-1 mutants. This approach could provide further insights into the role of SPL/NZZ in auxin-mediated pathways.\n8) Line 285: Clarifying the rationale for choosing IAA3/SHY2 as a candidate for testing the hypothesis, including its expression pattern during MMC differentiation, would strengthen the study’s focus and relevance.\n9) Line 292: I do not agree with the sentence “Even if with different percentages, in all the lines we observed an impairment in the MMC differentiation, resembling the spl-1 phenotype”. SHY2 transgenes are able to maintain the MMC formation at a significantly higher ratio than the spl-1 mutants, as shown in Figure 5M.\n10) The authors highlight ARF9 as a critical factor downstream of SPL/NZZ, yet ARF9 expression levels were found to increase in spl-1 mutants. The authors propose that increasing ARF9 in spl-1 backgrounds can rescue MMC differentiation, which logically conflicts with the observation of elevated ARF9 expression already present in spl-1 mutants. This discrepancy suggests a complex post-transcriptional or post-translational regulation not clearly explained, thus weakening the logic of ARF9’s functional role in MMC differentiation.\n11) PIN1 expression under the SPL/NZZ promoter in spl-1 mutants rescues MMC differentiation, the mechanism by which PIN1 re-expression bypasses other downstream consequences of SPL/NZZ loss remains unclear. For instance, integument defects were also rescued by PIN1 re-expression, yet funiculus elongation remained impaired. Clearer explanations for these differential rescue effects are required for logical consistency. Assessing whether PIN1 and ARF9 transgenes can restore MMC differentiation defects in MADS-domain TFs mutants, such as sep mutants, and whether common target genes shared by SPL/NZZ and MADS-domain TFs are recovered in both PIN1 and ARF9 re-expression, would provide insights into the interplay between these regulatory pathways.\nSome textual suggestions:\n1) Line 27: mutations in the SPL/NZZ gene lead to…\n2) Line 277: AUX/IAAs should be Aux/IAAs, in the whole manuscript.\n3) Line 340: a key step of the SPL/NZZ downstream…\n4) Line 399: carf17 mutant should be arf17 mutant?\n5) Line 428: PCM should be PMC?\n6) Figure 3M, remove the two dots above the box plot of wild-type data to enhance clarity.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "The mechanistic basis of female germ line formation in plants remains unclear. Thus, the authors address an important question of interest to scientists working in the fields of plant development and sexual reproduction.\nIn this paper, the authors investigate the formation of the megaspore mother cell (MMC). SPL/NZZ (SPL) is the earliest known factor controlling this process. In the past, work from several labs has shed some light on the function of SPL, but the genetic and molecular details of the SPL/NZZ downstream response have remained largely elusive. In this paper, the authors present a major advance in our understanding of the underlying mechanism and thus in our knowledge of early germline development.\nIn a first set of experiments using a variety of omics approaches (Co-IP/MS, ChIP-Seq, RNAseq) in part with the well-established pAP1::AP1:GR ap1cal1 system, the authors identify MADS domain transcription factors as preferred binding partners of SPL. The list of MADS TFs included well-known oocyte regulators such as STK, SEP3 and AG. Y2H assays suggested direct interaction of SPL with the three TFs, and expression studies confirmed the presence of the three TFs in young ovules. ChIP-Seq identified many direct targets of SPL. Interestingly, the authors found a large overlap with binding sites for SEP3. Comparison of RNA-seq data between wild type and spl-1 revealed that SPL promotes the activity of genes involved in e.g. chromatin remodeling and flower development, while SPL attenuates the expression of genes involved in auxin biosynthesis and signaling.\nIn a second set of experiments, prompted in part by previously obtained data, the authors focused on investigating the relationship between SPL and auxin signaling. Results from a complex set of genetic and cell biological experiments suggested that SPL mediates MMC formation by repressing ANT in the nucellus, which in turn allows for regular expression levels of PIN1 in the nucellus, accumulation of an auxin maximum at its distal tip, followed by relief of ARF9 repression by degradation of IAAs and MMC formation.\nI find the results presented in this paper highly interesting and of value for a general audience.\nComments:\n1. In my opinion the paper presents two stories that are only partially linked to each other. While the Co-IP/ChIP data are interesting their biological relevance is not addressed. For example, what is the role of one or several of the TFs interacting with SPL in MMC formation? In addition, the Y2H data suggest direct interaction between the tested TFs. However, Y2H data stem from a heterologous system and can be prone to artefacts. What about some tests (BiFC, FRET) in plant cells? While the RNA-seq data suggest that SPL is a general repressor of auxin processes, the authors make a link to the second story via the downregulation of PIN1 in spl-1. Thus, this connection appears somewhat artificial. The authors find binding of SPL/SEP3 to ANT regulatory sequences. In addition, they find upregulated ANT expression in the spl-1 dataset. Using these findings to make the link to PIN1 would make a more logical transition to the second part of the paper.\n2. There is a concern with the temporal sequence of events when it comes to the downregulation of PIN1 in spl-1. The authors report that the PIN1-GFP signal is unaffected until stage 1-II and note that it is reduced by stage 2-III. The presence of an MMC is clearly evident by stage 2-I and its formation has been proposed to occur even earlier (Hernandez-Lagana, et al. 2021 eLife 10, e66031). One would therefore expect PIN1-GFP to be reduced by late stage 1-II. This discrepancy needs to be explained by the authors.\nMinor comments:\nThere are several typos scattered throughout the manuscript and the supplement. Please fix.\nLegend to Figure S1. Nucellus is a Latin term. The plural is “nucelli” not “nucella”. Please fix.", "reviewer_4": "The formation of germline cells is fundamental in plants and animals. In Arabidopsis, the SPL/NZZ is required for the formation of both megaspore mother cell (MMC) and pollen mother cell (PMC). However, since its discovery in 1999, the molecular mechanisms by which SPL/NZZ controls MMC differentiation have remained poorly understood. In this study, the authors identified SPL/NZZ interacting proteins and direct target genes using omics approaches. The findings provide compelling evidence from multiple angles that SPL/NZZ specifies MMC fate by regulating auxin signaling, highlighting an important advancement in this field. Overall, the experiments were adequately designed and well executed. The data presented are extensive and beautiful. However, a major concern is the lack of genetic evidence supporting the roles of SEP and ANT genes in MMC specification.\nThe authors demonstrate that SPL/NZZ forms a regulatory complex together with MADS-box transcription factors, particularly the SEP3. While SEP3, STK, AG, and BEL1 are known to be involved in chalaza and integument development, the specific role of SEP3 in MMC formation remains unclear. The authors acknowledged that gene redundancy and pleiotropic effects make it difficult to assess the necessity of these MADS-box genes for MMC differentiation, but without direct genetic evidence, it needs to be cautious to conclude that “SPOROCYTELESS/NOZZLE acts together with MADS-domain transcription factors to regulate an auxin-dependent network controlling the Megaspore Mother Cell development.”\nTo circumvent some of the technical challenges, the authors might consider overexpressing SEP3 and conducting a conditional knockout of SEP genes using the SPL/NZZ promoter.\nThe identification of direct target genes of SPL/NZZ is a significant progress. Still, there is no genetic evidence supporting the function of ANT in MMC formation, despite the observation that ANT loss of function restores MMC specification in the spl mutant. It would be valuable to revisit MMC formation in ant mutants using molecular markers to understand how this restoration occurs. Is it possible that overexpression of ANT hinders MMC formation?\nAdditionally, do spl-1 pSPL/NZZ::PIN1 and spl-1 pSPL/NZZ::ARF9 plants produce seeds?\n“PCM” should be “PMC” in line 428, and “MCC” should be “MMC” in line 548."}, "author_response": "Point-by-Point responses to reviewer comments\nReviewer #1 (Remarks to the Author):\nThe manuscript by Cavalleri et al. investigates the role of SPL/NZZ in regulating\nmegaspore mother cell (MMC) development by identifying ovule-identity MADS-domain\ntranscription factors as direct targets, co-regulating an auxin-dependent downstream\nnetwork. SPL/NZZ is recognized as a master regulator in plant reproduction, particularly in\nthe differentiation of MMCs and PMCs. In this manuscript, the authors performed Co-\nIP/MS of SPL/NZZ and identified several MADS-domain transcription factors as SPL/NZZ-\nspecific interacting proteins during the early MMC stages of ovule development. The\nauthors then verified their interactions in yeast, supporting the formation of multimeric\nprotein complexes. ChIP-seq assays show that SPL/NZZ and the MADS-domain TFs\nshare common targets, over 90%, indicating that they might function in the same pathway\nto regulate the MMC development. Through Biological Process GO term enrichment\nanalysis done on these target genes, they made a direct link between SPL/NZZ and auxin.\nFurthermore, using loss-of-function mutants and complementation assays it is shown that\nthe severe developmental defects observed in the MMC differentiation in spl-1 mutant\novules could be due to the impairments in PIN1-mediated auxin transport and\naccumulation, and Aux/IAA-ARF module-mediated auxin transcriptional responses.\nTogether the multi-omics, genetic, and cell biological data conclusively demonstrate that\nSPL/NZZ acting with MADS-domain transcription factors determines the specification and\ndevelopment of the MMC via an auxin-dependent downstream network.\nIn general, the manuscript reads well and results are presented in appropriate figures.\nSeveral aspects warrant further clarification and additional experiments to strengthen the\nstudy’s claims.\n1) Line126: The authors used yeast-two- and three-hybrid assays to confirm the\ninteractions between SPL/NZZ and MADS-domain TFs. To provide more robust evidence\nof these interactions in their native context, fluorescence resonance energy transfer\n(FRET) or fluorescence lifetime imaging microscopy (FLIM-FRET) analyses in living plant\ncells, especially during MMC differentiation, are recommended.\nWe thank the reviewer for the comment. To support the conclusion that SPL not only form\na complex with SEP3 and STK, but that it also directly interacts in planta with these two TFs\nwe performed, as suggested by the reviewer, FRET-FLIM using tobacco leaves as\nexpression system. The SPL interacting partners have been identified by a Co-IP/MS\nexperiment, using flower synchronized just before and at MMC differentiation stages.\nThanks to the suggestion of the reviewer, we are more confidents to conclude that STK and\nSEP3 non only form a complex with SPL, but they do directly interact during MMC\nspecification. The GFP was used as donor protein, and we fused it to either SEP3Δ or STK\nc-terminus. By contrast, the mCHERRY was selected as acceptor and it was fused to SPLΔ\nc-terminus. FLIM measurements revealed a significant reduction in the GFP lifetime when\nSEP3Δ-GFP or STK-GFP were co-expressed with SPLΔ-mCHERRY in tobacco nuclei,\nconfirming SEP3 and STK ability to physically interact with SPL. The results of this\nexperiment are presented in Figure1, in the Supplementary Figure 3 and in the main texts\nat lines <149-160>.\n2) Line 153: The manuscript references Alphafold predictions to suggest the formation of\nSPL/NZZ-SEPs-STK complexes. To substantiate these predictions, detailed docking site\nfeatures and docking scores should be presented, offering insights into the potential\nstructural configurations of these protein interactions.\nWe agree with the reviewer on the need of additional quantitative data regarding the\nprediction of the structure of protein complexes formed by SPL, SEPs and STK. Therefore,\nwe performed a binding affinity prediction of the SPL/NZZ-SEPs-STK complexes using the\nProdigy webserver. In all cases, we obtained Gibbs free energy and dissociation constant\nresults that validate the reliability of the Alphafold models. We also obtained a more detailed\nvisualisation of the SPL/NZZ-SEP-STK docking sites, highlighting the residues within the\nhydrogen bonds range (5.5Å). This information is presented in Figure 1, in Supplementary\nFigure 4 and in the main text from lines <170-177>\n3) Line 189: With over 90% overlap between SPL/NZZ and SEP3 ChIP-seq peaks, qRT-\nPCR assays should be conducted on representative target genes, particularly those involved\nin the ovule specification and development, to validate the ChIP-seq findings.\nWe thank the reviewer for the comment. To further dissect the activity of SPL/NZZ over its\ndirect targets, we analysed the SPL/NZZ-SEP3 common target list in the light of the DEGs\nobtained from the spl-1 RNA-seq. We found that 77 SPL/NZZ-SEP3 targets resulted also\ndifferentially expressed in the spl-1 pistil with respect to the wild type (Supplementary Data\n3). Nicely, these include genes involved in ovule development such as ANT, LUG, AP2,\nCUC3, IPT1, SEP3, AGL15 and FUL. Interestingly, all the auxin biosynthetic genes present\nin the spl-1 RNA-seq, including also the genes targeted directly by SPL/NZZ, resulted\nupregulated (Supplementary Data 3).\nWe described this analysis in the main text at line <252-259>.\n4) Line 219: Why did the authors choose to evaluate auxin transport, accumulation, and\nresponse concerning SPL/NZZ activity, but not auxin biosynthesis? The rationale for this is\nnot fully logical, as some data suggest that SPL/NZZ function is closely linked to auxin\nbiosynthesis. RNA-seq assay shows an impairment in auxin biosynthesis in the spl-1\nmutant. SPL/NZZ bound genes include the gene YUCCA6, which might be a direct target of\nSPL/NZZ, as later data show that SPL/NZZ-ANT module regulation over PIN1 is indirect in\novule development.\nWe agree that we need to add an explanation for the rationale behind the decision to focus\non auxin transport, accumulation, and response concerning SPL/NZZ activity, but not auxin\nbiosynthesis. We did not focus on auxin biosynthesis because it was previously described\nthat genes responsible for auxin biosynthesis are expressed in the chalaza region of the\novule, while SPL/NZZ function on MMC specification is played in the nucellus where its\nprotein is localized at that stage. This information is based on the expression pattern of key\nplayers in the auxin biosynthetic pathway as YUCCA 1, 2 3, 4, 8, 9 and TAA1 (as reviewed\nin https://doi.org/10.1093/jxb/erv256). Auxin is then conveyed to the ovule tip through its\ntransport in the L1 layer by PIN1, where SPL/NZZ localises. Moreover, it was previously\ndescribed that PIN1 expression and auxin response was downregulated in the spl mutant.\nWe also analysed in detail the expression of auxin biosynthetic genes in the spl-1 RNA-seq.\nAs described in the main text: <line 258> Interestingly, all the auxin biosynthetic genes\npresent in the spl-1 RNA-seq, including also the genes targeted directly by SPL/NZZ,\nresulted upregulated (Supplementary Data 3). Key players of auxin biosynthesis such as\nYUCCA 2, 4, 6 and TAA1, which are normally expressed in the ovule chalaza, showed\nenhanced expression. This result is in line with SPL/NZZ proposed activity as a\ntranscriptional repressor, which is also supported by the reduced expression of YUC2 and\n4 in the spl-D dominant mutant. After biosynthesis in the chalaza, auxin is polarly transported\ntowards the primordium apex mainly by PIN1, generating a region of high signalling output\nat the nucellus tip. Auxin is then directed toward the L2 layer by PIN3 and PIN1 action. Since\nit was already reported that PIN1 was drastically downregulated in spl-1 ovules, as well as\nthe auxin response, we decided to investigate in more detail auxin transport and response\nin wild-type and spl-1 ovules to evaluate their impact on spl-1 phenotype. <line 269>\nWe generated new tables, that we added to the Supplemental Data 3, to clearly highlight\nthe expression of auxin biosynthetic genes in spl-1 with respect to the wild type.\n5) Line 227: The signal of PIN1-GFP in wild-type and spl-1 ovules should be quantified\n(Figure 3A-D).\nWe thank the reviewer for the comment. We quantified the PIN1-GFP signal in the L1 layer\nof the nucellus in both wild type and spl-1 mutant. As we observed a different PIN1-GFP\nbehaviour at different timepoints between wild type and spl-1, we chose to focus on PIN1-\nGFP intensity at both stage 2-I and 2-III. We added also the analysis of DR5v2 signal at the\nsame time-points, together with the re-organisation of the corresponding paragraph.\nAs described in the main text: <line270> Focusing on wild type ovules, we observed that\nPIN1-GFP signal increased in the nucellus from stage 2-I, the stage in which the\ndifferentiation of the MMC initiates, to the stage 2-III, associated with the complete\ndifferentiation of the MMC (Figure 3A, B, M). Likewise, we measured a significant increase\nin the nucellar DR5v2 signal passing from stage 2-I to stage 2-III (Figure 3E, F, N). This\ndynamic in PIN1 and DR5v2 expression during the MMC differentiation could suggest the\nrequirement of auxin transport and signalling to support the MMC identity acquisition.\nIndeed, even though we observed that PIN1-GFP accumulated similarly in wild-type and\nspl-1 ovules at stage 2–I (Figure 3A, C, M), at stage 2-III, PIN1-GFP signal was drastically\nreduced in the spl-1 mutant (Figure 3B, D, M), as previously shown. Likewise, the DR5v2\nsignal observed between wild type ovules at stages 2-I and 2-III (Figure 3 E-H, N) was\nseverely reduced in in the spl-1 background. (Figure 3 E-H, N). Besides PIN1, also PIN3 is\ninvolved in auxin transport in the nucellus. Despite this, pPIN3::PIN3:GFP signal was\nlocalised at the very tip of ovule primordia in spl-1, and no differences were observed\ncompared to the wild type (Supplementary Figure 7A-D).\nDownregulation of PIN1 expression and DR5v2 response suggests that auxin is\naccumulated at lower levels in spl-1 nucellus. Indeed, by using the R2D2 reporter line, we\ndetected a higher accumulation of DII-VENUS in the spl-1 nucellus with respect to the wild\ntype (Figure 3I-L; Supplementary Figure 7E-I).\nConsidering the behaviour of PIN1 and DR5v2, together with the fact that auxin transport\nwas previously suggested to be required for the MMC specification, we believe that PIN1\ndownregulation could be the main cause of MMC absence in the spl-1 mutant ovule.\n<line290>\n6) Line 233: Investigating whether mutants of MADS-domain TFs, such as sep mutants,\nexhibit impaired auxin response similar to the spl-1 mutant could reveal functional overlaps\nand distinctions within this regulatory network.\nWe agree with the reviewer regarding the need of additional data to reveal functional\noverlaps between MADS-domain TFs and SPL/NZZ. <line211> Our data indicate that\nSPL/NZZ could require the MADS-domain TFs SEP1, 2, and 3, as well as STK and SHPs\nfor MMC specification. This outcome was also predictable, as it has been shown that MADS-\ndomain TFs of classes C, D and E form multimeric complexes to establish ovule tissue and\ncell identity. Regrettably, these transcription factors exhibit significant redundancy and\nmultiple mutants, as the shp1 shp2 stk ag/+ one, displays ovules homeotically changed into\nleaves, whereas the sep1 sep2 sep3 mutant has all floral organs homeotically changed into\nsepals. Therefore, to investigate the potential relevance of these MADS-domain TFs in MMC\ndifferentiation, we created a pSPL/NZZ::MADSas construct, to facilitate post-transcriptional\ngene silencing of SEP genes in the nucellus (Supplementary Figure 5A). We subsequently\nintroduced this construct into shp1/shp1 shp2/shp2 stk/STK plants, intending to inhibit the\nactivity of many ovule-identity MADS-domain TFs within the nucellus. We obtained three\nindependent transformant lines expressing the pSPL/NZZ::MADSas (Supplementary Figure\n5B). Specifically, lines 1 and 2 resulted shp1 shp2 STK/STK, whereas line 3 was classified\nas a triple shp1 shp2 stk mutant. Although the phenotype was much less penetrant with\nrespect to that reported in spl-1, the transgenic plants shp1 shp2 stk pSPL/NZZ::MADSas\nand shp1 shp2 STK/STK pSPL/NZZ::MADSas showed a ratio of 25% to 45% of ovules that\nfailed to produce the MMC (Figure 2E, F) besides an impairment in the organisation of the\nnucellus (Figure 2E; Supplementary Figure 5C).\nAltogether, these data support that SPL/NZZ and MADS-domain TFs cooperate, forming a\nmultimeric complex for the differentiation of the MMC. <line 231>\nWe think that these results could provide additional support regarding the cooperation\nbetween SPL and MADS during the MMC development. The results of this experiment can\nbe found in Figure 2, Supplementary Figure 5 and in the main text from <lines 211-231>.\n7) Line 265: The complementation assays demonstrating PIN1’s ability to rescue ovule\ndevelopmental defects in spl-1 mutants would benefit from the inclusion of negative controls,\nsuch as inactive versions of PIN1, to confirm the specificity of the observed rescue effects.\nAdditionally, it would be valuable to assess whether the application of exogenous auxin can\nrescue the developmental defects observed in the spl-1 mutants. This approach could\nprovide further insights into the role of SPL/NZZ in auxin-mediated pathways.\nThe spl-1 complementation by PIN1 expression is supported by multiple evidence. In first\ninstance, we analysed different independent lines, with the aim to avoid that the final\noutcome of the experiment could be altered by possible biases, as the position of insertion\nof the transgene in the genome. Secondly, we used the same SPL/NZZ regulatory\n regulatory\nsequences and expression system to drive the expression of other cDNAs sequences such\nas MP one, without obtaining a consistent rescue of spl phenotype. Therefore, we are\nconfident that the SPL/NZZ regulatory sequences in the spl-1 background does not, by itself,\ncomplement the spl phenotype.\nConcerning the application of exogenous auxin to rescue the MMC development in the spl-\n1 mutant, we have done the experiments obtaining negative results. Inflorescences were\ntreated with 100µM IAA and collected for DIC microscopy after 24h from the treatment.\nDespite this, no differences were observed between mock and treated spl-1 ovules. We\ndon’t believe that these results will add useful information to the manuscript since, we think\nthat the problem in the spl mutant is the incapability to reach a threshold in auxin\nconcentration in specific nucellar cells due to the severe downregulation of PIN1.\n8) Line 285: Clarifying the rationale for choosing IAA3/SHY2 as a candidate for testing the\nhypothesis, including its expression pattern during MMC differentiation, would strengthen\nthe study’s focus and relevance.\nWe thank the reviewer for the comment, and we are sorry for the lack of clarity. The main objective\nof the experiment was to impair the MMC differentiation by repressing the ARFs activity. To do so\nwe thought to took advantage of the constitutive inhibitory features of an Aux/IAA dominant mutant\nvariant. We described in more detail the rational of our choice in the main text as follow: <line352>\nSingle-cell RNA-seq, performed using ovules between stages 2-I and 2-III, revealed\nthat Aux/IAAs 1, 2, 3, 4, 8, 9, 13, 16 and 26 were expressed in nucellar clusters. To select\nthe appropriate Aux/IAA to repress the ARFs activity in the nucellus, we chose the one that\nwas previously reported to interact with several ARFs expressed in the nucellus, and for\nwhich was available a dominant auxin-resistant allele. We choose the Aux/IAA3/SHY2\ndominant mutant allele, named shy2.6. shy2.6 carries a mutation in the degron motif,\nimpairing its ability to interact with the SCF/TIR1 complex at threshold auxin level, remaining\nconstitutively bound to ARFs PB1 domain. Consequently, we generate the\npSPL/NZZ::shy2.6 construct to express shy2.6 in the nucellus of wild-type ovules. <line361>\n9) Line 292: I do not agree with the sentence “Even if with different percentages, in all the\nlines we observed an impairment in the MMC differentiation, resembling the spl-1\nphenotype”. SHY2 transgenes are able to maintain the MMC formation at a significantly\nhigher ratio than the spl-1 mutants, as shown in Figure 5M.\nWe are sorry for the misleading phrasing and thank the reviewer for the observation. We\nmodified the main text as follow: <line363> Even if with a lower penetrance with respect to\nthe spl-1 mutant, in all the lines we observed an impairment in the MMC differentiation,\nresembling the spl-1 phenotype (Figure 5A-M). <line364>\n10) The authors highlight ARF9 as a critical factor downstream of SPL/NZZ, yet ARF9\nexpression levels were found to increase in spl-1 mutants. The authors propose that\nincreasing ARF9 in spl-1 backgrounds can rescue MMC differentiation, which logically\nconflicts with the observation of elevated ARF9 expression already present in spl-1 mutants.\nThis discrepancy suggests a complex post-transcriptional or post-translational regulation not\nclearly explained, thus weakening the logic of ARF9’s functional role in MMC differentiation.\nWe thank the reviewer for the comment. We totally agree that the description related to\nARF9 expression has to be rephrased. Indeed, ARF9 is expressed in ovule, as shown by\nthe ISH, in both wild type and spl. Despite this, in the spl-1 RNA-seq, we observed a\ndecrease in ARF9 expression level in the spl-1 pistil. This finding corroborates, as reviewer\n1 suggested, that the activity of ARF9 may be regulated by complex post-translational\nregulations. Indeed, if ARF9 activity depends on an Aux/IAAs-dependent post-translational\nregulation, increase the amount of ARF9 protein in the nucellus at the right stages is\nsupposed to rescue, at least partially, spl mutant phenotype, as we have observed. We\nagree with the reviewer1 that this concept has to be better explained and commented. We\nmodified the main text to increase the clarity of this point <line 391-399>.\n11) PIN1 expression under the SPL/NZZ promoter in spl-1 mutants rescues MMC\ndifferentiation, the mechanism by which PIN1 re-expression bypasses other downstream\nconsequences of SPL/NZZ loss remains unclear. For instance, integument defects were\nalso rescued by PIN1 re-expression, yet funiculus elongation remained impaired. Clearer\nexplanations for these differential rescue effects are required for logical consistency.\nThank you for the suggestion. We have modified the text and added additional experiments\nto better explain the different rescue defects.\nRegarding the rescue of integuments development in spl-1 pSPL/NZZ:PIN1 ovules we\ndiscussed as follow: <line327> ovules could correctly differentiate inner and outer\ninteguments (Figure 4G-J, L-O). Complementation of inner integuments development\nsuggests that SPL/NZZ is important for the proper PIN1 localisation and auxin transport for\ninner integuments. Indeed, regulation of auxin transport was shown to be involved in\ninteguments formation and differentiation <line330>.\nConcerning the funiculus length, <line332> We hypothesised that this phenotype could be\nrelated to the fact that genes involved in funiculus differentiation remained deregulated in\nspl-1 pSPL/NZZ::PIN1 ovules. For instance, it was shown that ANT ectopic expression is\ndirectly associated with the differentiation of a longer funiculus. By ISH, we observed that\nANT remained ectopically expressed in both nucellus and funiculus of spl-1\npSPL/NZZ::PIN1 ovules (Supplementary Figure 10C). The ectopic expression of ANT in spl-\n1 pSPL/NZZ::PIN1 not only provide a hierarchical view in which PIN1 is placed downstream\nANT but also represent a plausible explanation for the elongated funiculus of spl-1\npSPL/NZZ::PIN1 ovules. <line340>\nAssessing whether PIN1 and ARF9 transgenes can restore MMC differentiation defects in\nMADS-domain TFs mutants, such as sep mutants, and whether common target genes\nshared by SPL/NZZ and MADS-domain TFs are recovered in both PIN1 and ARF9 re-\nexpression, would provide insights into the interplay between these regulatory pathways.\nWe agreed with reviewer 1 that the use of PIN1 and ARF9 constructs to rescue the MMC\ndifferentiation in sep mutants would provide insights into the interplay between these\nregulatory pathways. However, it is important to consider that the multiple sep mutants\nde sep mutants\ndevelop only sepals instead of flower organs while the complete loss of ovule identity in the\nstk shp2, shp1 ag/+ background determines the conversion of ovules into leaves. Thanks to\nthe pSPL/NZZ::MADSas lines, we provided evidence that directly links the MADS function\nto the MMC formation. Despite this, we could only achieve a partial loss of MMC\ndifferentiation, making the analysis of the potential effect of PIN1 and ARF9 on MMC\ndevelopment in these lines difficult to evaluate. We discussed the fact that SPL action is,\nprobably, mediated by multiple redundant MADS <line 441-457>. Likewise, we discussed\nthat understanding ARF9, and ARFs in general, mode of action and interplay with the other\ncomponents of the MMC developmental pathway will require additional work that will\nrepresent a completely new research topic <line 545-550>.\nSome textual suggestions:\n1) Line 27: mutations in the SPL/NZZ gene lead to…\nWe modified as suggested\n2) Line 277: AUX/IAAs should be Aux/IAAs, in the whole manuscript.\nWe modified as suggested\n3) Line 340: a key step of the SPL/NZZ downstream…\nWe modified as suggested\n4) Line 399: carf17 mutant should be arf17 mutant?\nIt should be indeed carf17, as described in the manuscript in which this mutant is\npresented\n5) Line 428: PCM should be PMC?\nWe modified as suggested\n6) Figure 3M, remove the two dots above the box plot of wild-type data to enhance clarity.\nWe modified the Figure 3 introducing a new line graph to show PIN1-GFP intensity. The\nprevious 3M graph is now placed in the Supplementary Figure 7 and we removed the\noutlier points from the graph as suggested\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review and\nto provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nWe thank the reviewer for its evaluation of our manuscript\nReviewer #3 (Remarks to the Author):\nThe mechanistic basis of female germ line formation in plants remains unclear. Thus, the\nauthors address an important question of interest to scientists working in the fields of plant\ndevelopment and sexual reproduction.\nIn this paper, the authors investigate the formation of the megaspore mother cell (MMC).\nSPL/NZZ (SPL) is the earliest known factor controlling this process. In the past, work from\nseveral labs has shed some light on the function of SPL, but the genetic and molecular\ndetails of the SPL/NZZ downstream response have remained largely elusive. In this paper,\nthe authors present a major advance in our understanding of the underlying mechanism\nand thus in our knowledge of early germline development.\nIn a first set of experiments using a variety of omics approaches (Co-IP/MS, ChIP-Seq,\nRNAseq) in part with the well-established pAP1::AP1:GR ap1cal1 system, the authors\nidentify MADS domain transcription factors as preferred binding partners of SPL. The list\nof MADS TFs included well-known oocyte regulators such as STK, SEP3 and AG. Y2H\nassays suggested direct interaction of SPL with the three TFs, and expression studies\nconfirmed the presence of the three TFs in young ovules. ChIP-Seq identified many direct\ntargets of SPL. Interestingly, the authors found a large overlap with binding sites for SEP3.\nComparison of RNA-seq data between wild type and spl-1 revealed that SPL promotes the\nactivity of genes involved in e.g. chromatin remodeling and flower development, while SPL\nattenuates the expression of genes involved in auxin biosynthesis and signaling.\nIn a second set of experiments, prompted in part by previously obtained data, the authors\nfocused on investigating the relationship between SPL and auxin signaling. Results from a\ncomplex set of genetic and cell biological experiments\nsuggested that SPL mediates MMC formation by repressing ANT in the nucellus, which in turn allows for regular expression\nlevels of PIN1 in the nucellus, accumulation of an auxin maximum at its distal tip, followed\nby relief of ARF9 repression by degradation of IAAs and MMC formation.\nI find the results presented in this paper highly interesting and of value for a general\naudience.\nComments:\n1. In my opinion the paper presents two stories that are only partially linked to each other.\nWhile the Co-IP/ChIP data are interesting their biological relevance is not addressed. For\nexample, what is the role of one or several of the TFs interacting with SPL in MMC\nformation? In addition, the Y2H data suggest direct interaction between the tested TFs.\nHowever, Y2H data stem from a heterologous system and can be prone to artefacts. What\nabout some tests (BiFC, FRET) in plant cells?\nWe thank the reviewer for comment and suggestions. Even if the centrality of SPL/NZZ\naction during MMC development was well known since its first description, so far, its\nmolecular mode of action was still poorly understood. One of the greatest advances in this\nfield has been proposed by Wei and colleagues (2015), when they demonstrated that SPL\ndo not have a DNA binding domain and works as a corepressor of the transcription by\ninteracting with TFs.\nWith our work we confirm that SPL binding to the DNA depends on its recruitment into TF\ncomplexes. Indeed, we have identified that a large portion of SPL direct targets in ovules at\naround stage 2-I to stage 2-III, which are the stages from which we isolated SPL/NZZ-GFP\nto perform ChIP-seq (using synchronised inflorescences at 9DAI), are bound at the level of\nCarG box sequences, as a consequence of SPL interaction with MADS-domain TF\ncomplexes, at least at this stage of development.\nEven if many of SPL-interacting partners as SEP3, STK, SHPs and AG, were shown to be\nfundamental for ovule formation, their involvement in the MMC definition was not known.\nMoreover, the discovery of the involvement of these TFs in the germline specification could\nopen up new line of research, as their study during the male germline formation.\nConcerning the generation of additional data to confirm the interaction between SPL and\nfactors as SEP3 and STK in-planta, we performed FRET-FLIM using tobacco leaves as\nexpression system as suggested also by reviewer 1. The GFP was used as donor protein,\nand we fused it to either SEP3Δ or STK c-terminus. By contrast, the mCHERRY was\nselected as acceptor and it was fused to SPLΔ c-terminus. FLIM measurements revealed a\nsignificant reduction in the GFP lifetime when SEP3Δ-GFP or STK-GFP were co-expressed\nwith SPLΔ-mCHERRY in tobacco nuclei, confirming SEP3, STK ability to physically interact\nwith SPL. The results of this experiment are presented in Figure1, in the Supplementary\nFigure 3 and in the main texts at lines <149-160>.\nWhile the RNA-seq data suggest that SPL is a general repressor of auxin processes, the\nauthors make a link to the second story via the downregulation of PIN1 in spl-1. Thus, this\nconnection appears somewhat artificial. The authors find binding of SPL/SEP3 to ANT\nregulatory sequences. In addition, they find upregulated ANT expression in the spl-1\ndataset. Using these findings to make the link to PIN1 would make a more logical transition\nto the second part of the paper.\nWe thank the reviewer, and we are sorry if the rationale behind the link between SPL and\nthe auxin signalling has appeared uncertain. To understand the biological pathway\nassociated with SPL/NZZ activity our first approach has been based on the analysis of the\nGO categories enriched in our omics datasets. Ontology analysis pointed out the connection\nbetween SPL/NZZ and the auxin signalling. This association has not been unexpected, as\nprevious works already pointed out the requirement of auxin transport and response for the\nMMC differentiation. Likewise, the downregulation of PIN1 in the spl mutant background was\nalready described. After having clarified whether PIN1 downregulation could have been a\ncrucial factor associated with the absence of the MMC in the spl ovule, our logical approach\nhas been to investigate a possible molecular mechanisms that could link SPL/NZZ to PIN1.\nThe absence of PIN1 among SPL/NZZ direct targets, together with its downregulation in the\nmutant ovule, clearly indicated the existence of one or more intermediate that connect\nSPL/NZZ to PIN1. Starting from these finding, we thought to searched, among SPL direct\ntargets, factors which could be genetically associated with SPL, and which could work as\nintermediary of SPL/NZZ action over PIN1. Our attention was caught by ANT, especially\ndue to the existence of previous works reporting the ability of ant mutation to partially rescue\nthe MMC differentiation in the spl background.\nDespite this, no data connecting ANT activity to PIN1 expression were known, hindering the\ndirectly link between ANT ectopic expression to PIN1 miss-regulation in spl-1. Indeed, to\nmake this connection has required us testing whether ANT could repress PIN1. By ISH, we\nshow that ANT could indeed work as repressor of PIN1, as indicated by the expansion in\nPIN1 expression domain in the ant-4 mutant. We modified the main text <line 294-314> to\nenhance the clarity.\n2. There is a concern with the temporal sequence of events when it comes to the\ndownregulation of PIN1 in spl-1. The authors report that the PIN1-GFP signal is unaffected\nuntil stage 1-II and note that it is reduced by stage 2-III. The presence of an MMC is clearly\nevident by stage 2-I and its formation has been proposed to occur even earlier (Hernandez-\nLagana, et al. 2021 eLife 10, e66031). One would therefore expect PIN1-GFP to be reduced\nby late stage 1-II. This discrepancy needs to be explained by the authors.\nWe thank the reviewer for rising this very interesting point. As the reviewer suggested, the\nMMC starts its differentiation from stage 2-I. Despite this, the MMC differentiation is a\ndynamic process that span from stage 2-I to stage 2-III. Indeed, in this time frame, the MMC\nundergoes to important modifications. These include sensible enlargement in cellular and\nnuclear dimensions, the deposition of callose, and the expression of cellular specific\nmarkers. Indeed, some features as callose punctuation are visible only from stage 2-II and\nespecially at stage 2-III. According to this, the MMC differentiation has to be considered a\ndynamic process that initiates and conclude across different ovule developmental stages.\nAs shown by Hernandez-Lagana, et al. 2021, before the start of the MMC differentiation, in\nthe nucellus one or multiple potential MMC initial are present. <line500> Over time, the\nnucellar phenotypic plasticity is reduced until only one MMC precursor is present, at stage\n2-I. The plasticity in the identification of potential MMC candidates is also supported by the\neviction of the histone marker HTR13-GFP that, until stage 2-I, occurs in multiple nucellar\ncells. Eviction of HTR13-GFP in one single nucellar cell become prominent from stage 2-II,\nsupporting the clear differentiation of the MMC from the other cells. Despite this, it is\ninteresting to notice that wild type and spl-1 ovules, at precocious developmental stages,\npresent similar phenotypes allowing the morphological identification of one or multiple\npotential MMC candidate cells even in the mutant background (Supplementary Figure 16A-\nD). This suggest that, although the MMC do not differentiate, the formation of putative MMC\nprecursors is not affected in the spl-1 mutat (Supplementary Figure 16A-D). According to\nthis, the process of MMC development could be divided into two main steps: the first one\nwould be the formation of one or multiple MMC candidates, an event which could happen\nindependently from SPL/NZZ action. This step is limited by the reduction of the nucellar\nphenotypic plasticity toward the selection of one single MMC candidate that occur around\nstage 2-I. The second step would be the SPL/NZZ-dependent MMC identity acquisition,\nwhich starts from stage 2-I until MMC full differentiation and separation from the other\nnucellar cells at stage 2-III. According to this, SPL/NZZ action could be temporarily restricted\nduring the MMC identity acquisition phase, which correspond to the step in which we\nobserved the major changes in PIN1 expression between wild type and spl-1 ovules.\n<line519>\nWe discussed this point in the discussion section <line 491-525>, and we introduced an\nadditional Supplementary Figure (Supplementary Figure 16) to show wild type and spl-1\novules phenotype ad precocious developmental stages.\nMinor comments:\nThere are several typos scattered throughout the manuscript and the supplement. Please\nfix.\nThank you we fixed the typos\nLegend to Figure S1. Nucellus is a Latin term. The plural is “nucelli” not “nucella”. Please\nfix.\nThank you, we fixed the error\nReviewer #4 (Remarks to the Author):\nThe formation of germline cells is fundamental in plants and animals. In Arabidopsis, the\nSPL/NZZ is required for the formation of both megaspore mother cell (MMC) and pollen\nmother cell (PMC). However, since its discovery in 1999, the molecular mechanisms by\nwhich SPL/NZZ controls MMC differentiation have remained poorly understood. In this\nstudy, the authors identified SPL/NZZ interacting proteins and direct target genes using\nomics approaches. The findings provide compelling evidence from multiple angles that\nSPL/NZZ specifies MMC fate by regulating auxin signaling, highlighting an important\nadvancement in this field. Overall, the experiments were adequately designed and well\nexecuted. The data presented are extensive and beautiful. However, a major concern is\nthe lack of genetic evidence supporting the roles of SEP and ANT genes in MMC\nspecification.\nThe authors demonstrate that SPL/NZZ forms a regulatory complex together with MADS-\nbox transcription factors, particularly the SEP3. While SEP3, STK, AG, and BEL1 are\nknown to be involved in chalaza and integument development, the specific role of SEP3 in\nMMC formation remains unclear. The authors acknowledged that gene redundancy and\npleiotropic effects make it difficult to assess the necessity of these MADS-box genes for\nMMC differentiation, but without direct genetic evidence, it needs to be cautious to\nconclude that “SPOROCYTELESS/NOZZLE acts together with MADS-domain\ntranscription factors to regulate an auxin-dependent network controlling the Megaspore\nMother Cell development.” To circumvent some of the technical challenges, the authors\nmight consider overexpressing SEP3 and conducting a conditional knockout of SEP genes\nusing the SPL/NZZ promoter.\nWe thank the reviewer, and we agree with its evaluation. <line210> Our data indicate that\nSPL/NZZ could require the MADS-domain TFs SEP1, 2, and 3, as well as STK and SHPs\nfor MMC specification. This outcome was also predictable, as it has been shown that MADS-\ndomain TFs of classes C, D and E form multimeric complexes to establish ovule tissue and\ncell identity. Regrettably, these transcription factors exhibit significant redundancy and\nmultiple mutants, as the shp1 shp2 stk ag/+ one, displays ovules homeotically changed into\nleaves, whereas the sep1 sep2 sep3 mutant has all floral organs homeotically changed into\nsepals. Therefore, to investigate the potential relevance of these MADS-domain TFs in MMC\ndifferentiation, we created a pSPL/NZZ::MADSas construct, to facilitate post-transcriptional\ngene silencing of SEP genes in the nucellus (Supplementary Figure 5A). We subsequently\nintroduced this construct into shp1/shp1 shp2/shp2 stk/STK plants, intending to inhibit the\nactivity of many ovule-identity MADS-domain TFs within the nucellus. We obtained three\nindependent transformant lines expressing the pSPL/NZZ::MADSas (Supplementary Figure\n5B). Specifically, lines 1 and 2 resulted shp1 shp2 STK/STK, whereas line 3 was classified\nas a triple shp1 shp2 stk mutant. Although the phenotype was much less penetrant with\nrespect to that reported in spl-1, the transgenic plants shp1 shp2 stk pSPL/NZZ::MADSas\nand shp1 shp2 STK/STK pSPL/NZZ::MADSas showed a ratio of 25% to 45% of ovules that\nfailed to produce the MMC (Figure 2E, F) besides an impairment in the organisation of the\nnucellus (Figure 2E; Supplementary Figure 5C).\nAltogether, these data support that SPL/NZZ and MADS-domain TFs cooperate, forming a\nmultimeric complex for the differentiation of the MMC. <line231>\nThe results of this experiment can are show in Figure 2, Supplementary Figure 5 and in the\nmain text from <lines 211-231>.\nThe identification of direct target genes of SPL/NZZ is a significant progress. Still, there is\nno genetic evidence supporting the function of ANT in MMC formation, despite the\nobservation that ANT loss of function restores MMC specification in the spl mutant. It would\nbe valuable to revisit MMC formation in ant mutants using molecular markers to understand\nhow this restoration occurs. Is it possible that overexpression of ANT hinders MMC\nformation?\nWe observed that the MMC is specified in the ant mutant background and correctly divide\nthrough meiosis as was also previously reported (Losa et al., 2010). However, we agree\nwith the reviewer on the need of additional studies to clarify precisely how ANT regulates\nlate sporogenesis and gametogenesis. The main ant defects reported are the lack of\ngametogenesis and the impaired integuments initiation probably due to a miss regulation of\nPIN1 and hormonal homeostasis in the chalaza. This is a current line of our research. As\npoint out by the reviewer, the genetic association between ANT and SPL during the MMC\ndevelopment was already known since ant mutation could rescue partially the spl mutation.\nIn addition, we were able to highlight ANT ability to suppress the expression of PIN1. The\nrole of ANT in regulating PIN1 in ovules can be support by the data included in our article\nand the analysis of ant mutants that, likely due to ectopic expression of PIN1, fails to proper\naccumulate auxin to form the integuments (as lateral structure of the chalaza)\nNevertheless, to gather additional data on ANT relevance for the MMC specification, we\nfollowed the reviewer suggestion regarding its ectopic expression in the nucellus. <line482>\nTo define whether ANT expression in the nucellus was sufficient to control PIN1, we\ntransformed wild type plants with a pSPL/NZZ::ANT construct. Even though we obtained 3\nindependent lines expressing the construct (Supplementary Figure 15A), we did not observe\ndefect in the MMC development (Supplementary Figure15B, C). This result is not\nunexpected since, in spl/nzz mutants, several genes involved in the acquisition of chalaza\nidentity are ectopically expressed. Therefore, we can conclude that SPL/NZZ is required to\nrepress, in the nucellus, the genes required for chalaza specification and that, probably,\nANT is only one of the genes that must be silenced to have a proper nucellus identity\nacquisition. Up to date, no information regarding ANT interacting partner and mode of action\nis available. <line490>\nWe believe that this topic could represents a new and exciting field of study that we, or\npotentially other research groups, will follow in the future.\nAdditionally, do spl-1 pSPL/NZZ::PIN1 and spl-1 pSPL/NZZ::ARF9 plants produce seeds?\nThe plants are fertile and produce viable seeds. Despite this we did not perform a detailed\nanalysis of seed development and plant fertility as, we believe, these are both\ndevelopmental processes whose study goes beyond the scope of this work. This is a very\ninteresting topics that we are planning to study in the near future.\n“PCM” should be “PMC” in line 428, and “MCC” should be “MMC” in line 548.\nWe fixed the typos. Thank you for the comment.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nOverall, the authors have addressed most of the previous concerns. However, a few minor issues\nremain:\n1. The FLIM-FRET images in Figure 1 are of low quality, making them difficult to interpret. Please\nprovide higher-resolution images with proper controls.\nWe thank the reviewer for the comment. The FRET-FLIM images presented in Figure 1 are\ngenerated by the SymPhoTime 64 software during FLIM measurement. In these images, each pixel\nis coloured according to its fluorescent signal decay time, as measured by the SymPhoTime 64\nsoftware. Therefore, the structures visualised in such images could be indeed of difficult\ninterpretation. Therefore, based on reviewer 1 comment, we have included Figure 1D-H that show\nsingle-channel images showing GFP and mCHERRY signals in the different nuclei analysed during\nFLIM measurement. However, we believe that providing a visual representation of the change in the\nGFP lifetime in the different conditions (expression of SEP3-GFP and STK-GFP either alone or in\ncombination with SPL/NZZ-mCHERRY) could be important. Therefore, we moved the images\nshowing the GFP lifetime (highlighting the position of nuclei in the images) to the Supplementary\nFigure 3\n2. While the AlphaFold prediction offers structural insights, it is computational and not\nexperimentally validated. It would be more appropriate to move this figure to the supplemental\nsection.\nAs suggested by the reviewer, we moved all the data concerning the Alpha Fold predictions of\nSPL/NZZ-SEPs-STK complexes to the Supplementary Figure 4.\n3. Please indicate the sample size for each quantification and statistical analysis throughout the\nmanuscript.\nWe ensured that information regarding the sample size of all quantifications and statistical\n and statistical\nanalyses is properly provided in the legends or in the materials and methods section.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part\nof the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nReviewer #3 (Remarks to the Author):\nIn this revised version of the manuscript, the authors addressed my main comments in detail.\n1. They added FRET-FLIM data from tobacco experiments demonstrating the principal ability of\ndirect interaction between SPL and SEP3 or SPL and STK in a plant cell. A test in Arabidopsis\nwould have been preferable but this may have been technically challenging.\nWe thank the reviewer to considering the FRET-FLIM experiment in tobacco sufficient to prove the\ndirect interaction of SPL, STK and SEP3 in planta.\n2. In my opinion, the author's response to my concern about the temporal sequence of events\nregarding the down regulation of PIN1 in spl-1 has been inadequate. My reasonings are as follows:\n- I am confused about how the authors use the terms \"identity acquisition/specification\" and\n\"differentiation,\" as these terms refer to two fundamentally different processes. I am also unclear\nabout the exact roles that the authors propose for SPL and PIN1 in these processes. In the\nabstract, the authors mention that SPL is a principal regulator of MMC formation because its\nmutant phenotype affects MMC differentiation. Later, they continue to highlight the role of a\nregulatory network involving SPL/PIN1 in controlling MMC specification, yet they also discuss the\nrole of SPL/PIN1 in MMC differentiation. I suppose the confusion stems from the question of\nwhether the SPL/ANT/PIN1 axis is involved in specifying the MMC or its differentiation following\nspecification, or in both processes. What is the authors's notion? Please clarify.\nWe thank the reviewer, and we agree that we must use more precisely the words 'specification' and\n'differentiation'; therefore, we have corrected the text in accordance with the previously published\nmanuscripts and our results.\nSPL action has been previously associated with the MMC differentiation. Indeed, several works such\nas Wei-Cai Yang 1999 and Wei 2015, describe that spl mutants do form an archespore, which does\nnot differentiate into the MMC. Therefore, we agree to use the same terminology used in the\npreviously published manuscript.\nOur genetic data, along with previous studies, have shown that both the ant mutation\n(https://doi.org/10.1242/dev.127.19.4227) and the pSPL::PIN1 construct can rescue the spl mutant\nphenotype; therefore, we have adopted the same terminology throughout the text as previously\nmentioned, following the suggestion of reviewer 3.\n- At the onset of stage 2-I, a single large MMC is clearly visible in the wild type. Once the single\nMMC is enlarged further MMC development takes place during subsequent stages. The authors\nagree that the specification of a single MMC must occur before stage 2-I. The single large MMC is\nmissing in spl-1. Based on cell morphology, the argument that MMC-like precursor cells appear\nduring stages 0-III or 1-I in both the wild type and spl-1 mutant is not convincing (Supplemental\nFigure 16). The larger cells present in the prospective nucellar region during stages 0/1 may or\nmay not be MMC precursors. They could be cells that are dividing but have not yet undergone\ncytokinesis.\nIn any case, if SPL critically affects PIN-1 expression during MMC specification, it must also occur\nbefore stage 2-I. Otherwise, MMC specification depends on other SPL-regulated factors, and\nSPL/ANT-regulated PIN1 expression is relevant for MMC differentiation following specification. Do\nyou suggest that a single MMC is specified in spl-1 but does not enlarge and continue\ndevelopment?\nYes, as previously mentioned, we have agreed that, as described before, SPL is required for MMC\ndifferentiation rather than specification.\nFailure in MMC enlargement/further differentiation could be explained by a role for\nSPL/ANT/PIN1/auxin in growth control (via for example auxin-mediated cell wall changes) and/or\npolarity control in the MMC. If so the text should clarify that specification of the single MMC is not\napparently affected by SPL.\nAs we have written above in the response to the reviewer 3 's previous comment, we have agreed\nthat the SPL-ANT-PIN1 action should be placed during the MMC differentiation phase, which\noccurs after MMC specification. We have changed the text accordantly. According to the timing of\nPIN1 deregulation, we agree with the reviewer that the PIN1 downregulation in spl-1 is of pivotal\nimportance to MMC differentiation and that the acquisition of MMC identity likely occurs at stage 2-I\nor even before. We properly described in the text the distinction in the SPL/ANT/PIN1 involvement\nin MMC differentiation with respect to the previous stages (line 502-521).\n- There are also conflicting reports in the literature. The Colombo lab published data revealing that\nectopic expression of SPL result in supernumerary MMCs (Mendes et.al. 2020 Development\n147:dev194274). At the same time, other previous papers from the Colombo lab have provided\nevidence that, upon amiRNA-based downregulation of PIN1 in young ovules, in the pin1-5 mutant,\nor in a cytokinin receptor mutant carrying ovules with no detectable PIN1pro:PIN1-GFP expression,\nearly development of the embryo sac is blocked in the vast majority of ovules (Bencivenga et al.,\n2012, Plant Cell, 24:2886; Ceccato et al., 2013, PLoS ONE, 8:e66148). Thus, these data do not\nsupport a role for PIN1 in MMC specification. Additionally, under such conditions, MMC\ndifferentiation and meiosis would not be impaired to the extent that meiosis and megaspore\nformation could not be completed. How can these findings be reconciled with a model in c control\nMMC specification?\nThe model in which SPL/ANT/PIN1 control MMC differentiation summarised genetic data\ndescribed in our manuscript and in previously published ones. We could complement the spl lack\nof a differentiated MMC by introducing PIN1 under the SPL promoter. We obtained similar results\nalso using the pSPL::ARF9 construct. By contrast, expressing pSPL::SHY2-6 in wild type ovules\nimpaired MMC differentiation significantly, even if both SPL and PIN1 resulted still expressed.\nTherefore, we are confident that the differentiation of MMC required a proper auxin signalling\nresponse. The role of ANT in this process is based mainly on genetic data provided by previous\nworks (for instance, the publication https://doi.org/10.1242/dev.127.19.4227) and on the role of\nANT in repressing PIN1.\nRegarding MMC differentiation in the mutant backgrounds cited by the reviewer, it is worth noting\nthat pin1-5 is a weak pin1 allele. In this mutant allele, listils are formed and contain ovules, which\nare completely absent in pin1 knock-out mutants. This suggests that a residual function of PIN1 is\nstill present in pin1-5 plants, which could be sufficient to support MMC differentiation. Concerning\nthe analysis of the amiRNA knock-down line of PIN1, as previously shown, those plants present a\nreduction of PIN1 expression of around 50% with respect to a wild-type situation, implying that\nPIN1 could still be functional during MMC development. Nevertheless, even if the focus on these\ntwo manuscripts was to study the overall ovule development, as reported in Bencivenga et al.,\n2012, around 10 % of pin1-5 ovule primordia were arrested as finger-like structures in which a\nclear MMC was not visible. The same was reported for cre1-12 ahk2-2 ahk3-3 ovules (comparison\nof figure 2c and 2e Bencivenga et al 2012).\nMinor point:\nThere are numerous typos scattered throughout the manuscript. Please correct them.\nWe have corrected the typos throughout the manuscript\nReviewer #4 (Remarks to the Author):\nThis resubmission includes a substantial amount of new data and revisions that address the\nreviewer’s main concerns, particularly the genetic evidence supporting the role of SEP genes in\nMMC formation. The new result shows that the ectopic expression of ANT in the nucellus does not\naffect MMC formation. The authors propose that ANT functions redundantly with other genes to\nspecify the nucellus identity. An earlier study showed that loss-of-function of ANT only partially\nrestored MMC formation in spl mutant, and these MMCs failed to develop into embryo sacs. Thus,\nit is possible that ANT is not the sole direct target of SPL in inhibiting PIN1 activity.\nThe working models in Figure 7 are somewhat misleading. Furthermore, the authors should\ndiscuss how ANT and other genes controlling nucellus identity, together with other target genes\ncontribute to MMC differentiation. Are pSPL/NZZ::ANT plants capable of producing functional\nembryo sacs or achieving normal seed set?\nThank you very much for the comment. We totally agree with reviewer 4 about the model\npresented in Figure 7. We apologise for the inaccuracy of the model in the previous manuscript\nversion.\nIt has been reported that the ant mutation partially rescued the MMC differentiation defect in spl\nmutants (https://doi.org/10.1242/dev.127.19.4227); however, our experiments indicated that ANT is\nlikely not the only direct SPL target involved in nucellus and MMC differentiation. Indeed, when we\nectopically expressed ANT in the nucellus, we did not observe any issues with MMC differentiation.\nTherefore, there are other factors, not expressed in the nucellus in wild type, which remain\nunknown to date, that could work together or in parallel with ANT to impair MMC differentiation\nwhen expressed in the nucellus (as in the spl mutant). According to our data, the SPL-dependent\nrepression of such factors should contribute to the establishment of the nucellar auxin response,\nwhich is central for MMC differentiation. The repression of chalazal genes could act on the\nspatiotemporal expression of PIN1. Indeed, besides ANT, BEL1 was also reported to be involved\nin PIN1 repression. Concerning BEL1, no information regarding its direct target genes is available\nto date. We discussed this point in the discussion section. <line 486-498>.\nRegarding pSPL/NZZ::ANT plants, the majority of the T1 independent lines analysed successfully\nproduced seeds, even if we noticed a reduction in the final seed production."}]

Decision letter