PM: Plasma Membrane; SI: Self-incompatibility; ROS: Reactive Oxygen Species; H2 O2 : Hydrogen Peroxide; FER: FERONIA; RALF: RAPID ALKALINIZATION FACTOR; RBOH: RESPIRATORY BURST OXIDASE HOMOLOG; PCP: POLLEN COAT PROTEIN; FC: Fold Change; RLK: Receptor Like Kinase; nFPKM: Normalized Fragments Per Kilobase of exon per Million reads mapped; HPCA1: HYDROGEN-PEROXIDE-INDUCED Ca2+ INCREASES; CRK: CYSTEINE-RICH RECEPTOR-LIKE KINASES. SRK: S-LOCUS RECEPTOR KINASE; SCR: S-LOCUS CYSTEINE-RICH PROTEIN

How sexual partners choose each other is greatly dependent on organisms, but this choice reveals particularly critical in angiosperms with predominantly sessile lifestyle and hermaphroditic flowers that greatly favors self-fertilization. Self/non-self-recognition mechanisms, known as SI systems, have evolved to prevent self-fertilization, hence promoting genetic variability [1]. In the Brassicaceae family, self/nonself-discrimination occurs when the male gametophyte (pollen grain), lands at the receptive surface of the female organ (stigma epidermis of the pistil). Compatible pollen (non-self-pollen) hydrate and germinate a pollen tube that penetrates the stigma epidermis and navigates within the pistil to transport the male gametes towards the ovules for fertilization. By contrast, self-pollen (also named incompatible pollen) is recognized through the interaction of the stigma plasma membrane (PM)-localized S-LOCUS RECEPTOR KINASE (SRK) and its cognate peptide ligand from the pollen surface, named the S-LOCUS CYSTEINE RICH PROTEIN (SCR) [2]. This recognition event triggers activation of a signaling cascade in stigmatic cells culminating in self-pollen inhibition. Indeed, accurate communication is required to precisely coordinate the stigmatic response, to promote compatible pollen whereas ensuring self-pollen rejection. A significant advance in the understanding of the molecular dialogue established between pollen grains and the stigmatic cells comes from two recent exciting studies. Liu et al. [3] identified an autocrine signaling pathway that controls basal production of ROS in mature Arabidopsis stigma. Two Receptor Like Kinases (RLKs) (FERONIA, FER and ANJEA, ANJ) belonging to the CrRLK1L (CATHARANTHUS ROSEUS RECEPTOR LIKE KINASE 1-LIKE PROTEINS) family, expressed in the stigma, sense the stigmatic peptide ligand RAPID ALKALINIZATION FACTOR 33 (RALF33). Ligand recognition in turn activates the plant NADPH oxidase, RESPIRATORY BURST OXIDASE HOMOLOG D (RBOHD), a PM-localized ROS?producing enzyme. The authors also demonstrated that peptide ligands from the POLLEN COAT PROTEIN B-CLASS (PCP-Bs), present at the surface of the pollen grain, compete with RALF33 for FER/ ANJ receptor binding, thereby reducing RBOH-dependent ROS production to facilitate compatible pollen hydration. Zhang et al. [4] confirmed the existence of a FER/RBOH signaling module responsible for the basal ROS production in the stigma of the SI species Brassica rapa. In addition, the authors demonstrated that this module mediated a rapid ROS increase upon self-pollination resulting in the early inhibition of the self-pollen. Recently, we developed an experimental procedure, coupled with a bioinformatic analysis, to comprehensively unravel the dynamic events that occur both in the stigma and pollen grain following compatible and incompatible pollinations [5]. Here, we reexamine these transcriptomic data in light of the recent advances in the field, mainly focusing on redox regulatory network and receptor-ligand complexes.

Transcriptomic design Detailed procedure is described in the original article [5]. Briefly, using two transgenic Arabidopsis lines with restored SI response [6], we performed a time-course experiment of pollination and sequenced mRNAs extracted from stigmas harvested immediately (zero time point, C0), 10 or 60 minutes after pollen addition. We took advantage of the Single Nucleotide Polymorphism existing between two distinct Arabidopsis thaliana accessions, to differentiate pollen and stigma transcripts. Differential expression analysis of the whole transcriptome was performed using DESeq2. Then, we analyzed the relative abundance of each transcript using normalized Fragments Per Kilo base of exon per Million reads mapped (nFPKM) and retained genes expressed in mature stigma or pollen (at zero time point), those with a nFPKM >1 (Supplementary Table 1). We examined the transcriptomic changes in response to compatible (C10 and C60) and incompatible (I10 and I60) pollinations using the Fold Change (FC) calculation and retained genes exclusively upregulated (FC>2, padj < 0.1) in one pollination condition compared to the other (compatible vs incompatible and vice versa), in stigma and pollen (Supplementary Table 2).

Gene list construction

 A list of 323 Arabidopsis Redox genes was retrieved from Oliveira et al. [7] (Supplementary Table 1). A list comprising 472 genes encoding Arabidopsis RLKs was retrieved from Lee and Goring [8]. From our bibliographic analysis, we found 11 additional putative RLKs. We analyzed these 11 sequences with the Simple Modular Architecture Research Tool; two of them do not have obvious transmembrane domain and one encodes a truncated WAKlike kinase with a very short extracellular domain. These three sequences were not included in the RLK list and we ended up with a list containing 480 RLK genes (Supplementary Table 1). Lease and Walker [9,10] described around 1000 putative peptides in Arabidopsis. In these studies, to allow some tolerance, the upper size limit was fixed at 250 amino acids based on the largest known signaling molecule, the tomato systemin precursor. Since 2010, a large number of studies has described the role of plant peptides in cell-tocell communication. Thus, to update the Lease and Walker peptide database, we compiled published data together with information from The Arabidopsis Information Resource (TAIR) to generate a list of 958 signaling peptide/small protein genes based on the same criteria as those used by Lease and Walker (Supplementary Table 1).

 Identification of Redox, RLK and peptide genes in our transcriptomic data

 We screened our stigma and pollen transcriptomes with the Redox, RLK and Peptide lists using the Excel VLOOKUP function and made a Venn diagrams (https://bioinfogp.cnb. csic.es/tools/venny/) to represent the results.

Stigmatic redox regulatory network ROS, such as hydrogen peroxide (H2 O2 ) and superoxide anion, are chemical compounds formed from oxygen. They are toxic byproducts of all aerobic organisms but at the same time they function as signaling molecules. Plants have a complex redox system, which comprises 323 antioxidant and ROSgenerating enzymes to maintain redox homeostasis [7]. To better characterize the stigmatic redox network, we screened our transcriptomic data from the mature stigma (at zero time point) with the list of 323 plant redox genes. As the stigma is the epidermal tissue of the pistil organ, we decided to compare its redox network with that of another epidermis, obtained from laser microdissected leaves (RNAseq data retrieved from Berkowitz et al., [11]). Interestingly similar number of genes are expressed in both epidermal tissues, 14,220 genes in stigma (nFPKM>1) and 13,707 in leaf (Transcripts Per Million, TPM>=1) (Supplementary Table 1). We found 160 redox genes expressed in stigma and 156 in leaf (Figure 1, Supplementary Table 2). Although the number of redox genes expressed in both epidermis is equivalent, we found considerably more redox genes among the upmost expressed genes in stigma (four genes among the top12, Figure 2). Interestingly, three of them (PER39, 2,528.79 nFPKM; PER9, 1,625.48 nFPKM; PER58, 1,569.97 nFPKM; in comparison the highest stigma-expressed gene has an abundance of 13,047.31 nFPKM, Supplementary Table 2) belong to the same gene family, the plant specific class III peroxidase family. Plant PM-bound RBOHs are responsible for the major source of ROS production in the apoplast [12]. In Arabidopsis, there are 10 RBOH isoforms, referred to as RBOHA to RBOHJ [13]. We found RBOHD to be the highest expressed RBOH gene in mature stigma with a relative abundance of 340.48 (Supplementary Table 2). Two additional RBOH genes were expressed in mature stigma: RBOHF (16.70 nFPKM) and RBOHC (1.27 nFPKM). None of these three RBOH genes was expressed in mature pollen, whereas two other RBOH isoforms, RBOHH (45.99 nFPKM) and RBOHJ (10.56 nFPKM) were identified in our pollen transcriptome (Supplementary Table 2). These data are in accordance with previous studies where the RBOHD was identified as the most abundantly expressed RBOH gene in the stigma [3] and RBOHH and RBOHJ were found as expressed in mature tricellular pollen [14,15]. Identification of candidate RLKs for putative stigmatic functions 240 RLK genes were found within the 14,220 stigmaexpressed genes and two RLKs linked to ROS sensing and signaling were among the highest RLK expressed genes (Supplementary Table 2, Figure 3): FER (180.02 nFPKM) and HYDROGEN-PEROXIDE-INDUCED Ca2+ INCREASES (HPCA1, 72.4 nFPKM)We found 26 RLK genes specifically upregulated upon compatible pollination, whereas only seven were induced after the incompatible reaction (Supplementary Table 3). Interestingly, among the seven incompatible-induced RLK genes, two belong to the CYSTEINE-RICH RECEPTOR-LIKE KINASES family (CRK31 and CRK41), whose several members are involved in the regulation of defense reactions and ROS signaling [16]. Both CRK31 and CRK41 were strongly upregulated upon incompatible pollination with FCs around nine (Figure 3). This FC corresponds to a substantial induction as the highest FC for stigmatic genes is 12 and only two genes exhibited a greater FC upon pollination than those of the two CRKs. It is worth noting that neither FER nor HPCA1 were upregulated upon pollination (Supplementary Table 3).

Signaling peptide and pollen-stigma interaction

We generated a list of 958 peptide/small protein genes (Supplementary Table 1). Their expression products are categorized into two classes: secreted and non-secreted peptides based on the presence of a N-terminal secretory signal sequence (Figure 4). Secreted peptides can be further divided into two major classes: CYSTEINE-RICH PEPTIDES that contain 4–16 cysteine residues necessary for the formation of intramolecular disulfide bonds, and post-translationally modified peptides that contain at least one modification such as tyrosine sulfation, proline hydroxylation, or hydroxyproline arabinosylation. Cell-to-cell signaling is mediated largely by secreted peptides, but there are also evidences that nonsecreted peptides, released from damaged cells, or acting intracellularly, could support signaling functions. Each peptide We identified 124 and 54 peptide genes in mature stigma and pollen, respectively (Figure 5, Supplementary Table 2). Based on database screening and quantitative RT-PCR analysis, Liu et al. [3] determined that RALF33, a FER ligand implicated in ROS production, was expressed in stigma. Likewise, we found RALF33 (96.91 nFPKM) to be the second highest expressed RALF gene in the stigma, RALF8 (109.46 nFPKM) being the upmost expressed one. We identified a multitude of RALF genes expressed in pollen. RALF9 (10,273.85 nFPKM) and RALF8 (9,471.26 nFPKM) were the fifth and sixth upmost expressed pollen genes respectively (Supplementary Table 2). In addition to RALF8 and RALF9, RALF26 (2,049.09 nFPKM), RALF4 (1,717.38 nFPKM) and RALF19 (1,484.03 nFPKM) were among the top12 expressed peptide genes in pollen (Figure 5, Supplementary Table 2). RALF13, RALF36, RALF6 and RALF7 were upregulated upon compatible pollination (Supplementary Table 3). Although a function in reproduction has been described for two of them (RALF4 and RALF19), the role of other pollen RALFs has not been reported yet. Pollen peptides from the PCP B-class regulate the establishment of pollen-stigma compatibility through interaction with the FER receptor to reduce stigma ROS [3,21]. RNA-gel blot analysis and in situ hybridization showed that four A. thaliana PCP-B encoding genes are expressed in pollen at late stage of development. Surprisingly, we did not detect any expression of the PCP B-class genes (including the 12 PCPB-like genes) in our pollen transcriptomes at zero time point or after pollination (Supplementary Tables 2 and 3).

Maintenance of redox homeostasis in stigmatic cells

 Class III peroxidases belong to a large multigenic family of 73 members in Arabidopsis [22], which all possess a signal peptide targeting the proteins into the secretory pathway [23]. Eight class III peroxidases are predicted to be PMlocalized [23], among them we found the stigma-expressed gene PER58. Class III peroxidases, as other peroxidases, catalyze the oxidation of a variety of substrates by consuming hydrogen peroxide (H2 O2 ), and indeed participate in the detoxification of ROS. It is now well established that stigma from angiosperms accumulate a large amount of ROS, believed to be associated with protection of the reproductive structures against pathogen [3,24,25]. Considering the activity of class III peroxidases in ROS detoxification, we may postulate that the three highly expressed isoforms, PER9, PER39 and PER58, participate in protecting the stigmatic cells against oxidative damages caused by ROS excess. However, depending on the oxidized substrate, class III peroxidases can also provide ROS molecules from H2 O2 and thus participate in the generation of ROS. Indeed, a ROS burst triggers upon pathogen attack is dependent on both the RBOHD enzyme and two Arabidopsis class III peroxidases, PER33 and PER34 [26]. Thereby, we may alternatively postulate that the three class III peroxidases highly expressed in the mature stigma, contribute to the basal level of stigmatic ROS in collaboration with the stigma-expressed RBOHD. Likewise, PER9, PER39 and PER58 might be part of the redox network responsive for the rapid and high induction of ROS levels to reject self-pollen. It is still not clear yet what are the determining factors that control peroxidases activity either as H2 O2 scavengers or ROS producers. Understanding the role of class III peroxidases in stigma function and elucidating how their opposite activities are coordinated remains   

ROS burst in SI response depends on RLK signaling

 We found two RLKs (HPCA1 and FER) linked to ROS sensing and signaling among the highest expressed RLK genes in stigma. HPCA1 belongs to the Leucine Rich Repeat subfamily and harbors a unique extracellular domain containing two cysteine pairs named the hydrogen peroxide domain. Activation of HPCA1 by H2 O2 occurs via covalent modification of the cysteine residues, which leads to HPCA1 autophosphorylation and subsequently activation of Ca2+ channels in guard cells [27]. Interestingly, self-pollination in SI Brassicaceae plants induces an increase in cytoplasmic Ca2+ in papilla cells mediated by GLUTAMATE-GATED-Ca2+ channels [28]. FER has emerged as a multifunctional regulator of diverse biological processes such as fertilization, cell growth, plant immunity, mechano-sensing and many of these processes require ROS production [29]. FER has two extracellular carbohydratebinding (lectin) domains and, in addition to sense peptide ligands from the RALF and PCP-B families, FER binds to pectin wall components [30]. FER maintains a high ROS environment in stigmatic cells and stimulates ROS overproduction that allows rejection of self-pollen [3,4]. Similarly, FER is required for high ROS production in response to the bacterial flagellin. Flagellin induces association of its cognate RLK, FLAGELLIN SENSING 2 (FLS2), with the co-receptor BRASSINOSTEROID INSENSITIVE 1–ASSOCIATED RECEPTOR KINASE 1 (BAK1); formation of this immune complex is FER-dependent [31] and triggers activation of the RBOHD enzyme that enhances ROS production [32]. We identified two RLK genes (CRK31 and CRK41) highly and specifically upregulated in the stigma after incompatible pollination. The extracellular domain of CRKs (44 family members, [33]) contains two cysteine-rich domains that correspond to DUF26 motifs (Cys-X8-Cys-X2-Cys). The molecular function of the CRK ectodomain remains unknown, it could either bind a ligand or be the target for redox regulation through cysteine modifications. Several studies highlighted the central role of CRKs in both biotic and abiotic stress responses. Recently, Kimura and collaborators [35] showed that CRK2 interacts and directly phosphorylates the plant RBOHD oxidase to generate a ROS burst essential to counteract pathogen infection. CRK36 also regulates ROS production interacting with and phosphorylating the RECEPTOR-LIKE CYTOPLASMIC KINASE BOTRYTIS-INDUCED KINASE (BIK1), a downstream effector of FLS2, that phosphorylates RBOHD [32,35,36]. In Addition, several CRKs associate with the FLS2 immune complex [37,38] suggesting that CRKs might act in concert with other RLKs. Beside their function in immune responses, CRKs have also been implicated in drought and salt tolerances [39,40]. In the Brassicaceae, self-pollen rejection clearly depends on the activation of a RBOH-dependent ROS production [4]. Moreover, the specificity of the SI response relies on the recognition of the incompatible pollen by the highly polymorphic stigmatic receptor SRK. It is tempting to speculate that cell surface ROS sensors we highlighted in this study, HPCA1 and/or CRK31/CRK41, might be part of the ROS-mediated SI response and serve to connect pollen recognition and ROS burst. In a first scenario, we propose that CRK31/CRK41 are phosphorylated by SRK following SCR perception. Activated CRKs then stimulate ROS production, by phosphorylating, directly or indirectly, RBOH enzymes. In a second scenario, SCR-SRK interaction triggers an initial ROS production in the apoplast. Apoplastic ROS are then sensed by HPCA1, CRK31 and/or CRK41 that in turn phosphorylate RBOHs, leading to a boost in ROS levels responsible for selfpollen inhibition.

Our work identified potential new molecular players to deeper understand the dialogue between the pollen grains and the stigmatic epidermis. Class III peroxidases might finetune ROS levels at the stigma surface and hence play an essential role in controlling ROS homeostasis in the mature stigma and/or mediating pollen discrimination during SI response. Self-pollen recognition relies on the specific interaction between the two highly polymorphic SRK and SCR proteins. A major question is how this initial step is connected to ROS production? We identified two RLKs (CRK31 and CRK41) highly induced upon incompatible pollination, known to activate RBOH enzymes and interact with other RLKs, as suitable candidates to link initial SRK activation and ROS burst during SI response. Whether SRK directly phosphorylates and activates CRK31/CRK41 and how FER fits in this signaling pathway remain elusive and required further investigations.

We thank Dr. Thierry Gaude for critical reading of the manuscript. This work was supported by the Centre National de la Recherche Scientifique and grant ANR-14-CE11-0021

CK was responsible for all experiments and analysis performed in the original study. IFL designed the study, and performed additional analysis presented in this manuscript. CK and IFL wrote the manuscript and approved the final manuscript.

All the generated data are included in this article

The Authors declare that they have no competing interests.