Molecular mechanisms of self-incompatibility in Brassicaceae and Solanaceae

Abstract

Self-incompatibility (SI) is a mechanism for preventing self-fertilization in flowering plants. SI is controlled by a single S-locus with multiple haplotypes (S-haplotypes). When the pistil and pollen share the same S-haplotype, the pollen is recognized as self and rejected by the pistil. This review introduces our research on Brassicaceae and Solanaceae SI systems to identify the S-determinants encoded at the S-locus and uncover the mechanisms of self/nonself-discrimination and pollen rejection. The recognition mechanisms of SI systems differ between these families. A self-recognition system is adopted by Brassicaceae, whereas a collaborative nonself-recognition system is used by Solanaceae. Work by our group and subsequent studies indicate that plants have evolved diverse SI systems.

Introduction

Most flowering plants are hermaphrodites (∼90%), with both male and female organs present in each flower.¹⁾ Because self-pollination occurs easily in these bisexual flowers, flowering plants have evolved systems to avoid self-fertilization and ensure genetic diversity within the species. Self-incompatibility (SI) is a major system and has been confirmed in 71 families and 250 genera of angiosperms (estimated to be approximately 60% of all species).²⁾ SI is generally controlled by a single locus called S, which consists of numerous alleles identified by numbers (S₁, S₂, …, S_n), although in some cases, two or more loci are involved in SI. The S-locus is a complex of at least two multi-allelic genes; these S-alleles are more accurately referred to as S-haplotypes by analogy with the major histocompatibility complex in animals.³⁾ Pollen rejection occurs when pollen and the pistil share the same S-haplotypes. In many species with homomorphic SI, such as the Brassicaceae and Solanaceae species addressed in this review, 10–40 S-haplotypes are often found in a single population.⁴⁾

Traditionally, SI is genetically categorized as sporophytic or gametophytic SI based on the phenotypic behavior of pollen. In gametophytic SI, the SI phenotype of the pollen is determined by its own haploid S-haplotype. In contrast, the SI phenotype of the pollen in sporophytic SI is conferred by the two S-haplotypes of the diploid plant (2n) rather than the S-haplotype present in an individual pollen grain (n). Thus, when a pollen parent has S₁- and S₂-haplotypes, the pollen grains exhibit an S₁- or S₂-phenotype in gametophytic SI, whereas all pollen grains generally exhibit both S₁- and S₂-phenotypes in sporophytic SI.

To explain these genetic controls of SI, each S-haplotype is expected to encode at least two SI phenotypic determinants working at the pollen and the pistil sides, and discrimination of self and nonself is enabled by a specific molecular interaction between these S-determinants. For the past 40 years, molecular studies of plant SI have progressed extensively in Brassicaceae, Solanaceae, and Papaveraceae. The molecular types and mechanisms of these SI systems are entirely different; an overview of the research on these systems is provided in several reviews.^5)–9) Here, we review our research on the identification of S-determinants and the elucidation of self/nonself-discrimination mechanisms in the Brassicaceae and Solanaceae families.

Self-incompatibility in Brassicaceae

The Brassicaceae family includes many common vegetables such as the Japanese favorites Chinese cabbage, cabbage, broccoli, and radish (daikon). Most of these vegetables are now derived from F₁ hybrid seeds produced by crossing two genetically different inbred lines using the property of SI. F₁ hybrid lines generally exhibit homogeneous and early growing phenotypes due to heterosis, underscoring the importance of SI in agriculture. Although Brassicaceae SI is sporophytically controlled by a single S-locus, it often shows dominant/recessive relationships between S-haplotypes, particularly on the pollen side. For example, in an S_aS_b heterozygote, the pollen exhibits both S_a- and S_b-phenotypes irrespective of the pollen’s own genotype, whereas if the S_a-haplotype is dominant over the S_b-haplotype, the pollen exhibits only the S_a-phenotype.

The top of the pistil (stigma) is covered with a single layer of cylinder-like cells called “papilla” (Fig. 1A). When a pollen grain lands on the papilla cell of the nonself (compatible) plant, the pollen absorbs water from the papilla cell and germinates, and the pollen tube penetrates the papilla cell. The pollen tube elongates through the cell wall, style transmitting tract, and finally reaches the ovule for fertilization. However, in self (incompatible) pollination, the pollen fails to absorb water and does not germinate; if it germinates, the pollen tube cannot penetrate the papilla cell (Fig. 1B).

Fig. 1.

S-determinants in Brassicaceae SI. (A) Left, flower of B. rapa. Right, scanning electron microscope photograph of B. rapa stigma (gift from Dr. Iwano). (B) Observation of stigmas during self- and nonself-pollination using fluorescence microscopy. Pollen tubes are stained with aniline blue. (C) Domain structures of SLG, SRK, and SP11 proteins. SP, signal peptide; EGF, epidermal growth factor-like domain; PAN, PAN domain; TM, transmembrane region; HV, hypervariable region. (D) S-locus structures of B. rapa haplotypes. Arrows show the coding region of each gene including introns. Lines show haplotype-specific regions and dotted lines show conserved regions among haplotypes.

Search for Brassicaceae S-determinants

The first clue in the search for Brassicaceae S-determinants was obtained by immunochemical experiments. The presence of S-haplotype–specific antigens was detected in pistil extracts using antibodies generated against Brassica oleracea pistil homogenates with different S-haplotypes.¹⁰⁾ A stigmatic glycoprotein that was later called S-locus glycoprotein (SLG) showed different band patterns by isoelectric focusing depending on the S-haplotype.^11),12) We started SI research by purifying 150–300 µg of SLGs from 10,000 stigmas of B. rapa S₈-, S₉-, and S₁₂-haplotypes each and determined the amino acid sequences using a protein sequencer.^13),14) S₈-SLG is a 405 amino acid protein containing seven N-glycosylation sites that are modified with sugar chains comprising seven or eight sugars.^14),15) The amino acid sequences are similar among the three haplotypes, although they show some diversity, whereas the structures of the sugar chains are basically the same in the different S-haplotypes, suggesting that the amino acid differences are important for self/nonself-discrimination.^14),16) The amino acid sequence of SLG has a region that is partially similar to epidermal growth factor (EGF); however, this information was not sufficient to predict the biochemical function of SLG in SI.¹⁴⁾ Therefore, the whole region of SLG was termed the S-domain; later analysis revealed that SLG consists of two lectin, EGF-like, and PAN domains (Fig. 1C).^17),18) S₈-SLG contains an additional 31 amino acids that form a signal sequence at the N-terminus of the cDNA and the protein localizes to the cell wall of papilla cells.^19),20)

In 1990, the first plant receptor kinase, ZmPK1, was cloned from maize.²¹⁾ Surprisingly, the extracellular domain of ZmPK1 showed similarity to SLG, suggesting that a class of receptor kinases with an S-domain–like extracellular region exists in plants (Fig. 1C).²¹⁾ A search for a receptor kinase with an S-domain–like extracellular region in B. oleracea identified a receptor kinase encoded on the S-locus named S-receptor kinase (SRK).²²⁾ We independently cloned SRK genes from B. rapa S₈-, S₉-, and S₁₂-haplotypes, which were shown to co-segregate with their cognate S-haplotypes.^23),24) The extracellular domain of these SRKs shares 66–77% sequence identity among haplotypes and 76–97% against the cognate SLG.²⁴⁾ SRK mRNA expression was detected in the stigma, suggesting that both SLG and SRK act on the pistil side.²³⁾ Introduction of an S₈-SLG antisense construct into B. rapa resulted in the breakdown of SI, suggesting that SLG, SRK, or both are important for SI function on the pistil side.^25),26) As a final proof of their function, S₂₈-SLG or S₂₈-SRK genes were introduced into S₆₀S₆₀-homozygous plants, and only the pistil of the S₂₈-SRK transformants acquired S₂₈-specificity, suggesting that the S-determinant on the pistil side is SRK and not SLG.²⁷⁾ However, the simultaneous introduction of S₂₈-SLG and S₂₈-SRK resulted in an even stronger rejection of cognate S₂₈-pollen, indicating that SLG functions in the SI reaction through an unknown mechanism.²⁷⁾

The identification of the pistil S-determinant as a receptor kinase suggested that the pollen S-determinant is its ligand. We analyzed the SLG/SRK genomic region of the S₉-haplotype of B. rapa and found a candidate ligand-encoding gene, which was designated SP11 (S-locus protein 11).²⁷⁾ The S₉-SP11 gene is located between the S₉-SLG and S₉-SRK genes and was specifically expressed in the anther; it encodes a small cysteine-rich basic protein.²⁸⁾ Schopfer et al. also reported an anther-expressed cysteine-rich protein that was independently named S locus cysteine-rich protein (SCR) and shown to be the pollen S-determinant by transgenic experiments in B. oleracea.²⁹⁾ We also identified the S₈-SP11/SCR (hereafter called SP11 to avoid confusion) gene as an S₈-haplotype–specific anther-expressed gene in a parallel experiment comparing anther cDNAs from S₈S₈- and S₁₂S₁₂-homozygotes of B. rapa using fluorescent differential display.³⁰⁾ We confirmed that these SP11s are pollen S-determinants using bioassay methods in addition to transformation experiments.^30),31) Treatment of recombinant SP11 protein on the stigma surface induced an S-haplotype–specific SI reaction, suggesting that SP11 is the pollen S-determinant of Brassicaceae SI.³⁰⁾

The amino acid sequences of SP11 exhibited very low similarities among S-haplotypes except for the N-terminal signal sequences and eight conserved cysteine residues. Using primers designed based on conserved signal sequences, we cloned SP11 alleles from a number of S-haplotypes (Fig. 1C).^30),32) In situ hybridization experiments suggested that SP11 is expressed in anther tapetal cells, which are parental cells and form a single cell layer inside the anther locule.³¹⁾ Immunohistochemical observations suggested that SP11 is secreted from the anther tapetum onto the pollen surface, which is consistent with Brassicaceae exhibiting sporophytic SI.³³⁾

Genomic analysis of the S-locus in B. rapa revealed that the SLG, SRK, and SP11 genes are localized within 100 kb, although the order in which they are lined up and the direction of transcription sometimes differs among S-haplotypes; in addition, their nucleotide sequences showed little homology, including the intergenic regions (Fig. 1D).^28),30),34) These observations suggested that homologous recombination should be suppressed within the S-locus to avoid SI breakdown caused by recombination between pistil and pollen S-determinant genes.

Mechanism of self/nonself-discrimination in Brassicaceae self-incompatibility

To further investigate how pollen and pistil S-determinants discriminate self or nonself, we chemically synthesized mature (excluding signal sequence) regions of S₈- and S₉-SP11 using a redox reaction to form disulfide bonds. The mass of the synthetic S₈-SP11 was equal to that of intact S₈-SP11, indicating that the mature form of S₈-SP11 has four disulfide bonds.³⁵⁾ S₈-SP11 did not bind to the extracellular domain of S₈-SRK in surface plasmon resonance experiments, probably due to immobilization of S₈-SRK in its monomeric form. However, radio-labeled S₈-SP11 specifically bound to the stigma microsomal membrane of the S₈-haplotype but not of the S₉-haplotype.³⁵⁾ Scatchard analysis indicated that there are two SP11 binding sites in microsomal membranes, with dissociation constant (K_d) values of 0.7 and 250 nM.³⁵⁾ S₈-SP11 specifically bound 120- and 65-kDa proteins immunoprecipitated from the S₈ microsomal membrane using an SRK antibody but not to those from S₉, revealed that SP11 specifically binds to the cognate SRK.³⁵⁾ Detail liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis identified the 65-kDa protein as the truncated SRK protein including the extracellular domain and transmembrane region (tSRK).³⁶⁾ Membrane-anchored tSRK and dimerization domain–fusion SRK bound to SP11, but detergent-solubilized tSRK did not bind, suggesting that active dimer formation is important for high affinity binding.³⁶⁾ The SP11-induced haplotype-specific auto-phosphorylation of SRK established the story that the SP11 protein released from pollen specifically binds cognate SRK and activates the SRK kinase domain by inducing auto-phosphorylation, thereby triggering SI signaling.³⁵⁾

Next our question was how SRK discriminates self/nonself-SP11. SRK has three hypervariable (HV I–III) regions that share low amino acid similarity among haplotypes, supposing that the HV regions are involved in ligand recognition, and the amino acid variation enables the binding specificity of SRK against the cognate SP11 (Fig. 1C).³⁷⁾ However, predicting the SRK binding region of SP11 was difficult because mature SP11s have few conserved amino acids among S-haplotypes.^30),32) To address this question, we first determined the structure of S₈-SP11 using a nuclear magnetic resonance (NMR) approach.³⁸⁾ The solution structure of S₈-SP11 forms an α/β sandwich fold consisting of a single α-helix and three β-strands, which is a typical fold in defensin-like proteins (Fig. 2A).³⁸⁾ Homology modeling of other haplotypes based on S₈-SP11 suggested that the L1 and L2 loops of SP11s show structural diversity among haplotypes (Fig. 2A).³⁸⁾

Fig. 2.

Structures of S₈-SP11 and the S₈-SP11–S₈-SRK complex. (A) NMR structure of the SP11 monomer. Ribbons show α-helices and arrows show β-strands. N, N-terminus; C, C-terminus; L1, L1 loop; L2, L2 loop. (B) Crystal structure of the S₈-SP11–S₈-SRK heterotetramer complex. S₈-SRK molecules are shown in gray and cyan, and SP11 molecules are shown in orange and magenta. (C) Domain structure of SRK. Sugars and disulfide bonds are shown as a stick model. (D) HV regions on the SRK surface. SRK is shown as a surface model. HV regions are colored blue (HV I), green (HV II), and cyan (HV III). (E) Conservation profiles of the Brassica SRK protein. Conservation scores are indicated in color on the SRK surface. SP11 molecules are shown in yellow. Pictures are modified from Ref. 40.

To date, two SRK-SP11 complex structures have been determined using X-ray crystallography.^39),40) We determined the crystal structure of the S₈-SP11 and S₈-SRK extracellular domain complex as a heterotetramer consisting of 2:2 receptor and ligand molecules (Fig. 2B).⁴⁰⁾ In the S₈-SRK structure, two lectin domains are tandemly arranged, and the EGF and PAN domains associate with the second lectin domain (Fig. 2C). Three disordered sugar chains are observed, but they are not involved in ligand recognition. In a single S₈-SRK molecule, we found two ligand binding sites that are mainly composed of three HV regions (Fig. 2D). Two S₈-SP11 molecules are positioned between two S₈-SRK molecules oriented in a V shape and stabilize the homodimeric complex by bridging the two S₈-SRKs (Fig. 2B). Gel-filtration analysis showed ligand-induced SRK dimerization, suggesting that SP11 induces cognate SRK activation by promoting ligand-dependent dimerization.⁴⁰⁾ Mapping of sequence variability in 30 B. rapa SRKs on the structure revealed that amino acid residues around the SP11 binding pocket have high levels of diversity among S-haplotypes (Fig. 2E).⁴⁰⁾ Comprehensive binding analysis of 10 haplotypes of SRKs and SP11s with known structures or constructed by homology modeling by molecular dynamics (MD) simulation and molecular mechanics–generalized born surface area (MM–GBSA) calculations showed that SRK has high binding affinity for cognate SP11.⁴⁰⁾ These haplotype-specific SRK-SP11 interactions enable the suppression of SRK heterodimer formation between different haplotypes and promote precise self/nonself-discrimination through cognate SRK homodimerization. From these results, we concluded that the mechanism of self/nonself-discrimination in Brassicaceae SI is based on “self-recognition”.

Downstream signaling in Brassicaceae self-incompatibility

The pollen rejection responses in Brassicaceae SI are triggered by the SRK–SP11 interaction at the papilla cell surface, although little was known about the mechanisms of intracellular signaling resulting in self-pollen rejection. One clue for addressing these mechanisms was provided by a self-compatible (SC) B. rapa variant, yellow sarson, which has a modifier (m) locus independent of the S-locus causing SC.⁴¹⁾ In mm plants, the breakdown of SI occurs on the pistil side, predicting that the m gene product acts downstream of SRK. A positional cloning approach to identify the responsible m gene revealed that the M gene encodes a membrane-anchored protein kinase (MLPK).⁴²⁾ Transient expression of MLPK in mm plants restored SI, and the mlpk protein derived from the m-locus lost its kinase activity, suggesting that MLPK is involved in SI signaling via phosphorylation.⁴²⁾ Recombinant SRK kinase domain phosphorylated MLPK protein in in vitro phosphorylation experiments, suggesting that one of the substrates of SRK is MLPK.⁴³⁾ Direct interaction between SRK and MLPK was confirmed by in vivo bimolecular fluorescent complementation analysis.⁴⁴⁾ The MLPK gene has two translation start sites, resulting in two gene products that possess different N-termini required for membrane anchoring. One has a myristoylation motif and was modified by tritium-labeled myristic acid in an in vitro translation experiment, and the other one has an N-terminus composed of hydrophobic amino acids and was predicted to be directly anchored in the membrane.⁴⁴⁾ Recent knockout experiments of MLPK in B. napus by a Chinese group showed the breakdown of SI on the pistil side, confirming the importance of MLPK in SI signaling in Brassica.⁴⁵⁾

Calcium ions (Ca²⁺) are a typical second messenger involved in signaling by multiple biotic and abiotic stimuli in plant cells.⁴⁶⁾ Examination of the pollen–papilla interaction in Brassica showed that the amount of calcium on the papilla cell surface is higher in compatible pollination than incompatible pollination.⁴⁷⁾ Cytosolic Ca²⁺ fluctuations are observed in SC Arabidopsis thaliana papilla cells during compatible pollination by live imaging using Yellow Cameleon.⁴⁸⁾ Visualization of actin through the expression of mTalin-GFP in B. rapa revealed that actin bundles accumulate under compatible pollen, whereas they depolymerize in incompatible pollination.⁴⁹⁾ We found that the pollen coat is enough to enhance the export of Ca²⁺ to the cell wall in papilla cells, suggesting that certain substances induce compatible responses in papilla cells to accept pollen of the same species.⁵⁰⁾ Furthermore, we found that the attachment of compatible pollen or pollen coat to papilla cells induced the expression of a Ca²⁺ pump gene, autoinhibited Ca²⁺-ATPase 13 (ACA13). The ACA13 protein was shown to accumulate at the pollen attachment site, and ACA13 knockout lines exhibited reduced Ca²⁺ export, as well as defects in compatible pollen germination.⁵⁰⁾

It has been shown that A. thaliana is an SC species that evolved through mutation of the SP11 or SRK gene, and introduction of these genes from the related SI species A. lyrata restored SI in A. thaliana (see later chapters).^51),52) We introduced the Ca²⁺ sensor protein Yellow Cameleon 3.60 into SI A. thaliana and monitored Ca²⁺ dynamics during the pollination process. Cytosolic Ca²⁺ concentration in the papilla cells was increasing near the pollen attachment site in incompatible pollination but not in compatible pollination.⁵³⁾ The Ca²⁺ increase was also observed in papilla cell protoplasts in response to the addition of cognate SP11, suggesting that SI signaling triggered by the SRK–SP11 interaction induces Ca²⁺ influx in papilla cells.⁵³⁾ Direct injection of Ca²⁺, but not potassium ions, into papilla cells inhibited pollen hydration, suggesting that Ca²⁺ increase is the major SI signaling pathway and sufficient to induce the SI response in Brassicaceae.⁵³⁾ A summary of our findings on SI responses in Brassicaceae is shown in Fig. 3.

Fig. 3.

Mechanism of SI signaling in Brassicaceae. SP11 released from the self-pollen specifically binds cognate SRK and induces dimerization of the SRK ectodomain and activation of the intracellular kinase domain. Phosphorylation relay from SRK to MLPK and unknown downstream molecules induce Ca²⁺ influx resulting in pollen rejection. In the case of nonself-pollination, the pollen receives water from the papillae cell and germinates a pollen tube. Subsequently, the pollen tube penetrates the cell wall of the papillae cell. ACA13 proteins transport cytoplasmic Ca²⁺ to the extracellular region for providing Ca²⁺ to the pollen tube.

Mechanism of dominance hierarchy in Brassicaceae self-incompatibility

Dominant/recessive relationships among S-haplotypes are often observed in sporophytic SI.⁵⁴⁾ Suppressing one of the two S-haplotypes decreases incompatible crosses, enabling efficient reproduction. Comprehensive pollination analysis of 24 B. rapa haplotypes showed that dominant/recessive relationships are frequent on the pollen side, but rare on the pistil side.⁵⁵⁾ When plants are heterozygous for class I and class II haplotypes, which are phylogenetic groups classified according to SLG and SRK sequence similarities, the SI phenotype of the class II haplotype is suppressed on the pollen side (Fig. 4A).⁵⁵⁾ Plants heterozygous for class I haplotypes usually exhibit a co-dominant phenotype, whereas class II heterozygotes follow a linear dominance hierarchy (S₄₄ > S₆₀ > S₄₀ > S₂₉).^27),55) Thus, pollen heterozygous for S₆₀ and class I haplotypes or for S₆₀ and S₄₄ does not exhibit the S₆₀ phenotype, whereas pollen heterozygous for S₆₀ and S₄₀ or for S₆₀ and S₂₉ only shows the S₆₀ phenotype because the S₄₀ or S₂₉ phenotype is suppressed. It was quite intriguing to understand such a complex mechanism.

Fig. 4.

Mechanism of dominance hierarchy in Brassicaceae SI. (A) Phylogenetic tree of B. rapa SRKs. SRK ectodomain sequences were used for tree construction. (B) Genome structure of the class I and class II S-loci. Arrows show the coding region of protein-coding genes including introns. Rectangles show small RNA-coding genes. Lines show haplotype-specific regions, and dotted lines show conserved regions among haplotypes. (C) Alignment of Smi small RNAs and SP11 promoters. The box indicates a highly homologous region between the Smi and SP11 promoters. Mismatched bases are shown in magenta. (D) Alignment of Smi2 and SP11 promoters. The boxes show the core segment of small RNAs. Mismatched bases are shown in magenta, and G:U pairs are shown in blue. Matched base values were calculated using the first 21 nucleotides of Smi2. The pictures shown in (C) and (D) are modified from Refs. 59 and 60, respectively.

To investigate the mechanism of dominance hierarchy in the pollen SI phenotype, we first searched for class II SP11 genes by chromosome walking on the S₆₀ S-locus region because the class II SP11 gene was unable to be amplified by PCR with primers designed for class I SP11. The S₆₀-SP11 gene was located approximately 7 kb upstream of S₆₀-SRK, and the primers generated from its signal sequence region can be used to amplify other SP11 genes from class II S-haplotypes.⁵⁶⁾ The amino acid sequences of class II SP11s are relatively conserved among class II S-haplotypes, whereas there is no similarity with class I SP11s except for the eight conserved cysteine residues.⁵⁵⁾ S₆₀-SP11 mRNA was detected in S₆₀S₆₀ homozygous plants, but not in S₆₀S₅₂ heterozygotes, suggesting that the dominant/recessive relationships of Brassica SI in the pollen side are regulated by SP11s at the mRNA level.⁵⁶⁾ Dominance hierarchy within class II S-haplotypes is also controlled by the suppression of recessive SP11 mRNA.⁵⁷⁾ To elucidate the mechanism of recessive SP11 mRNA suppression, we analyzed DNA methylation status, a form of epigenetic regulation of gene expression, for the SP11 genome. No obvious methylation of SP11 was detected in DNA prepared from entire anther tissues. When DNA was preferentially extracted from the anther tapetum, high DNA methylation was specifically observed in the SP11 promoter region of the recessive haplotype but not of the dominant haplotype.⁵⁸⁾ The methylation levels of SP11 promoter in leaves and anthers are basically low and specifically increased in tapetal cells just before the initiation of SP11 transcription.⁵⁸⁾

We predicted the involvement of small RNAs in the recessive haplotype-specific de novo methylation. An in silico search revealed the presence of an inverted repeat region in the S-locus of dominant class I haplotypes that contained a sequence similar to the class II SP11 promoter region (Fig. 4B).⁵⁹⁾ The region was actually transcribed in the anther tapetum, producing a 24-nucleotide small RNA named Smi. Smi from the class I haplotype exhibits high sequence homology with the methylated class II SP11 promoter region, with a mispair score below 5.5, an evaluation value to predict small RNA target. Surprisingly, Smi expression was detected not only in the class I haplotypes but also in several class II haplotypes, although they contain a mutation at base 10, which decreases the homology to the target (mispair score above 7.5; Fig. 4C). Introduction of a class I Smi genome fragment into class II homozygotes induced the suppression of class II SP11 expression, but not in the case of class II Smi, suggesting that Smi controls dominant/recessive relationships between class I and class II haplotypes.⁵⁹⁾

Regarding the dominant/recessive relationships within class II haplotypes, we found the involvement of another 24-nucleotide small RNA named Smi2, which is produced from class II specific inverted repeats (Fig. 4B).⁶⁰⁾ Smi2 was detected in all class II haplotypes but not in the most recessive S₂₉-haplotype, in which the precursor was transcribed from the inverted repeat region, whereas Smi2 was shown not to be processed out. Smi2 is polymorphic in sequence, and the dominant Smi2 always shows high homology to the recessive SP11 promoter sequence with a mispair score of 5.5 or less (Fig. 4D). For example, S₆₀-Smi2 exhibits high homology to the SP11 promoter sequences of the recessive S₄₀- and S₂₉-haplotypes (both with a mispair score of 3.5), but exhibits low homology to those of the dominant S₄₄-haplotype (mispair score of 7.0) and the self S₆₀-haplotype (mispair score of 6.5).⁶⁰⁾ Transformation experiments revealed that the introduced S₆₀-Smi2 precursor inverted repeat genome fragment suppressed SP11 expression in recessive S₄₀ and S₂₉ homozygotes but not in dominant S₄₄ and self S₆₀ homozygotes. Taken together, we proposed a model in which the five-layer linear dominance hierarchy (class I > S₄₄ > S₆₀ > S₄₀ > S₂₉) in B. rapa is explained by two small RNAs, Smi and Smi2. We also confirmed that the linear dominance hierarchy in the related species A. lyrata is controlled by small RNAs as in Brassica.⁶¹⁾

Evolution of self-compatibility in Brassicaceae

Selfing, which is synonymous with self-fertilization, is one of the frequent evolutionary events in flowering plants and has been studied extensively as a model of adaptive evolution.⁶²⁾ Although A. thaliana is an SC plant in the Brassicaceae family, its ancestor is thought to have shown SI similar to closely related SI species A. lyrata and A. halleri. In fact, the A. thaliana genome still contains a nonfunctional S-locus, and three diverged haplogroups, A, B, and C, have been identified to date, supposing that at least three evolutionary events changing from SI to SC have occurred.^63)–65) In haplogroup A, some accessions, including Wei-1, still retain a functional SRK, whereas a 213-bp inversion in the SP11 gene is conserved in all haplogroup A accessions. Moreover, when the inversion of SP11 was restored and expressed in Wei-1, the transformants acquired SI, suggesting that the SP11 mutation is one of the evolutionary events that triggered selfing in A. thaliana.^65),66)

Similarly, introduction of A. lyrata SP11 and SRK genes into certain A. thaliana accessions resulted in the acquisition of SI; however, for some unknown reason, stable SI was acquired in few accessions such as C24.⁵²⁾ We also first tried to introduce SI into the A. thaliana Col-0 accession because Col-0 is a widely used strain for plant research and its genome sequence and T-DNA insertion lines are easily available. However, unlike the C24 accession, Col-0 harboring A. lyrata SRKb and SP11b showed quite weak SI phenotype, as reported previously.⁵²⁾ Reciprocal cross experiments between Col-0 and C24 revealed that the SI phenotype in Col-0 is strong on the pollen side, whereas it is weak on the pistil side.⁶⁷⁾ F₁ plants from Col-0 and C24 crosses exhibited a weak SI phenotype on the pistil side, suggesting that the factor causing weak SI in Col-0 acts dominantly.⁶⁷⁾ The original S-locus of Col-0 belongs to haplogroup A corresponding to the functional A. halleri S₄-haplotype, and the SRKA gene was interrupted by an inverted repeat in the C-terminal intracellular region (Fig. 5).⁶⁷⁾ We found that this SRKA pseudogene is expressed in papilla cells, and small RNAs are produced from the inverted repeat region, and we speculated that the introduced SRKb is silenced in Col-0 by these small RNAs. In fact, introduction of the Col-0 SRKA pseudogene into C24 was shown to destabilize SI even in C24, and introduction of the SRKb gene with synonymous substitutions in the kinase domain was shown to confer stable SI.⁶⁷⁾

Fig. 5.

S-loci of SI A. halleri and SC A. thaliana haplotypes. The A. halleri S₄-haplotype and A. thaliana Col-0 and C24 accessions belong to the same haplogroup and share a common ancestor. In Col-0, the SP11 gene is inactivated by an inversion, and SRK is also inactivated by an inversion resulting in inverted repeats producing small RNAs. In the C24 accession, SP11 and the intracellular region of SRK are lost, and the extracellular region of SRK forms an inverted repeat producing small RNAs. Arrowheads show the region of the inverted repeat.

Surprisingly, even in C24 accession, another inverted repeat was found in the SRKA extracellular region. This extracellular region of SRKA only shares 73% nucleotide identity with SRKb, thus the small RNAs did not silence SRKb, although it suppressed pistil SI when crossed with Uk-3 and Wei accessions harboring SRKA (Fig. 5).⁶⁷⁾ Approximately 80% of the A. thaliana 1,083 accessions carry one of these two SRKA inverted repeats, suggesting that the dominant SI suppressors may contribute to the spread of SC throughout the population faster than the recessive SP11 mutation when SC is favored.^66),67)

Self-incompatibility in Solanaceae

Solanaceae is also an economically important plant family that includes tobacco, red pepper, potato, and tomato. Because of the gametophytic control in Solanaceae SI, pollen rejection occurs when the haplotype encoded in the haploid pollen genome is consistent with either one of the two haplotypes in the pistil. The top of the pistil in Solanaceae plants is covered by a wet exudate, and the pollen absorbs water, germinates, and the pollen tube penetrates the pistil (Fig. 6A and B). However, incompatible pollen tubes are arrested in the middle part of the pistil. These phenotypic and physiologic differences from Brassicaceae SI supposed that Solanaceae possesses a different molecular mechanism mediating SI. Later research revealed that the SI system in Solanaceae is conserved in Rosaceae, Plantaginaceae, and Rutaceae and may have evolved from the ancestor of eudicots.^68)–71)

Fig. 6.

Mechanism of Solanaceae SI. (A) Flower of Solanaceae plant petunia (P. hybrida). (B) Phenotypes of self- and nonself-pollination in petunia SI. (C) Collaborative nonself-recognition model in Solanaceae SI. Arrows indicate the haplotype of S-RNase that is recognized by each SLF gene product. (D) Model of Solanaceae SI. After self and nonself S-RNase molecules invade the pollen tube, nonself S-RNase molecules are recognized by one or more types of SCF^SLF and detoxified via polyubiquitylation, whereas self S-RNase molecules are not recognized by any type of SCF^SLF and can degrade pollen RNAs.

Identification of pollen factors in Solanaceae self-incompatibility

The first identified pistil S-determinant was obtained by cDNA cloning of a stylar glycoprotein from Nicotiana alata.⁶⁸⁾ The pistil factor was called S-RNase because the protein showed sequence similarity with the T2 family of ribonucleases (RNases) and had RNA-degrading activity.⁷²⁾ Degradation of ribosomal RNA was specifically observed in incompatible pollen penetrating the pistil, suggesting that S-RNase localized to the pistil cell wall somehow moves into the pollen tube and arrests pollen tube growth by inducing RNA degradation.⁷³⁾ Transgenic expression or knockdown of the S-RNase gene in N. alata and Petunia infulata demonstrated that S-RNase is a determinant of Solanaceae SI on the pistil side.^74),75) Because the growth of self-pollen tubes is suppressed by the toxicity of S-RNase, it had been proposed that the pollen determinant is an inhibitor of S-RNase activity.⁷⁶⁾

We chose P. hybrida grown on our university campus as a research model for Solanaceae SI. We found two heterozygous lines (S₅- and S₇-haplotypes), one was SI and the other one was SC. Although the determined cDNA sequences of S₅- and S₇-RNase from the two heterozygous lines were the same, we found that the SI line was diploid and the SC line was tetraploid.⁷⁷⁾ Although it was well known that breakdown of gametophytic SI is often observed when diploid plants change to tetraploid, we did not resolve the reason at this point. We next performed chromosome walking from a lambda phage clone encoding the S-RNase gene to search for candidate pollen S-determinants among adjacent genes. However, it was difficult to identify the neighboring clones because of highly repetitive sequences. Fluorescence in situ hybridization experiments revealed that the S-RNase gene is located in a highly repetitive centromere region.⁷⁸⁾

We next targeted the S-locus of a Rosaceae plant, Japanese apricot (Prunus mume) cv. Nanko harboring S₁- and S₇-haplotypes.⁷⁹⁾ Approximately 60 kb sequences of the S-locus in P. mume S₁- and S₇-haplotypes were constructed using cosmid clones, which revealed that 13 or 14 genes were encoded in these regions.⁸⁰⁾ Comparison of the S-locus between the S₁- and S₇-haplotypes identified three genes located on haplotype-specific regions: one was an S-RNase and the others were F-box genes named S-locus F-box (SLF) and SLF-like 1 (SLFL1).⁸⁰⁾ The SLF gene was first identified from the S-locus of Antirrhinum hispanicum in Plantaginaceae as a candidate pollen determinant.⁸¹⁾ Although two more SLF-like genes (SLFL2 and SLFL3) were identified in the P. mume S-locus, we thought that SLF is considered the likely pollen determinant because the other SLFL genes show low sequence diversity between haplotypes.⁸⁰⁾ The F-box protein, which belongs to a large protein family in the plant kingdom, forms the SCF (SKP1–CUL1–F-box–RBX1) E3 ubiquitin ligase complex as a substrate recognition subunit and catalyzes the ubiquitylation of target proteins to induce their proteasome-dependent degradation.^82),83) The function of SLF was predicted to be nonself-specific S-RNase removal by the ubiquitin-proteasome pathway.⁸¹⁾ Introduction of the S₂-SLF gene fragment into S₁S₁ plants by a US group induced an SC phenotype in Petunia, suggesting that the SLF gene is the pollen determinant.⁸⁴⁾

Self/nonself-discrimination in Solanaceae self-incompatibility

Despite the identification of pollen and pistil determinants from the S-locus in Solanaceae SI, how SLF recognizes and detoxifies nonself S-RNase whereas it cannot recognize self S-RNase remains unclear. It was pointed out that the unequal amino acid sequence diversity between S-RNase and SLF means the SLF gene arose later than S-RNase, which is inconsistent with the putative coevolution of S-determinants.⁸⁵⁾ We cloned SLF cDNA from S₅, S₇, S₉, and S₁₁ homozygous pollen in Petunia and found that the amino acid sequence of S₇-SLF is identical to the reported S₁₉-SLF sequence even though there was 45% identity in S-RNase, suggesting that these SLFs (designated SLF1) do not determine the specificity between S₇- and S₁₉-haplotypes.^86),87) These results led us to hypothesize that unknown SLF-like genes expressed in pollen are encoded at the Petunia S-locus, and we performed transcriptome analysis to identify them. We finally identified a total of 30 SLF-like transcripts categorized into six types (SLF1–6) using pollen transcriptome data from six S-haplotypes.⁸⁶⁾ Introduction of SLF1–3 genes into Petunia with different haplotype backgrounds showed SLF type-dependent breakdown of SI (Fig. 6C). Type-specific interactions between SLF and S-RNase were observed using pull-down experiments, suggesting that these specific interactions detoxify the S-RNase.⁸⁶⁾ From these results, we proposed a “collaborative nonself recognition” model, by which all haplotypes of S-RNase except the self are covered by subset recognition of each SLF to explain the mechanism of self/nonself-discrimination in Solanaceae SI.⁸⁶⁾

Our next questions extend to how many SLFs act in Solanaceae SI and how these SLFs avoid recognition of self S-RNase. To address these questions, we performed a comprehensive transcriptome analysis of eight Petunia haplotypes and revealed that each haplotype possesses 16–20 SLFs phylogenetically categorized into 18 types (SLF1–18).⁸⁸⁾ Our recognition analysis of 129 S-RNase and SLF combinations using transgenic plants revealed that 18.6% of the combinations are positive. The 16–20 SLF genes are theoretically sufficient to recognize the majority of S-RNase haplotypes if the recognition ratio is applicable to other SLFs.⁸⁸⁾ We also confirmed that some haplotypes lost the corresponding SLF genes which function to detoxify self S-RNase, and others have phylogenetically differed SLF sequences from major sequences of the same type SLFs, probably do not recognize the cognate S-RNase.⁸⁸⁾

Mechanism of pollen rejection in Solanaceae self-incompatibility

The amino acid sequences of SLFs predicted that they form an SCF complex that acts as ubiquitin E3 ligase. To determine whether SLF proteins form an SCF complex, we expressed FLAG-S₇-SLF2 in Petunia pollen and co-immunoprecipitated FLAG-S₇-SLF2 and its binding proteins from pollen extracts. We identified four proteins including FLAG-S₇-SLF2 in the co-immunoprecipitated fraction and detected CUL1 (named CUL1-P), SKP1 (SSK1), and RBX1 (PhRBX1) proteins by LC-MS/MS analysis, suggesting that S₇-SLF2 forms an SCF complex.⁸⁹⁾ In vitro ubiquitylation assays using FLAG-S₇-SLF2 purified from pollen extracts revealed that the SCF^S7-SLF2 complex polyubiquitinates S₉- and S₁₁-RNases, but not S₅- and S₇-RNases. This substrate specificity is consistent with the results of transgenic experiments suggesting that the SCF^S7-SLF2 complex specifically ubiquitinates the corresponding S-RNases.⁹⁰⁾ Suppression of CUL1-P in Petunia pollen inhibited the pollen tube growth in compatible pistils, supposing that SCF components are required for SLF function.⁹⁰⁾ A model of the SI mechanism in Solanaceae is shown in Fig. 6D. Both self and nonself S-RNases in the pistil cell wall invade the pollen tube, and nonself S-RNases are recognized by a series of SCF^SLF complexes and detoxified via polyubiquitylation. Self S-RNases probably degrade mRNA and rRNA, resulting in the inhibition of protein synthesis and pollen tube growth.

Concluding remarks

In two decades of research, we established the “self” and “collaborative nonself” recognition models in Brassicaceae and Solanaceae SI systems, respectively (Fig. 7). We found that the recognition systems in Brassicaceae and Solanaceae are different despite undergoing the same SI phenomenon. Our research suggested that flowering plants may possess different molecular mechanisms of SI. Although only one other SI system has been clearly determined as a “self” recognition system in Papaveraceae to date, candidate S-determination genes were found in Convolvulaceae, Asteraceae, and Poaceae, suggesting that different molecular species are used for SI in these plants.^91)–95) Analysis of S-locus genes in heteromorphic SI species of Primulaceae and Passifloraceae suggested that the molecules that directly discriminate self and nonself as in Brassicaceae and Solanaceae are probably not encoded in the S-locus.^96)–98) These observations also show the diversification of SI systems in flowering plants. Recent advances in sequencing technologies have enabled the analysis of the S-locus of plant species with unknown SI systems and help elucidate the mechanisms underlying the molecular diversity of plant SI systems. The research that began with SI has now expanded to intra- and inter-species incompatibility.^99),100) We hope that our research will provide a roadmap for subsequent studies targeting other SI mechanisms.

Fig. 7.

Models for “self” and “collaborative nonself” recognition systems in SI.

Acknowledgments

We thank all the members of Intercellular Communications Laboratory at Nara Institute of Science and Technology, K. Hinata and M. Watanabe laboratory at Tohoku University, and other collaborators for their contributions.

Notes

Contributed by Akira ISOGAI, M.J.A.; Edited by Tsuneyoshi KUROIWA, M.J.A.

Correspondence should be addressed to: A. Isogai, Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan (e-mail: [e-mail]).

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Profile

Kohji Murase was born in Gifu Prefecture, Japan, in 1975 and graduated from Shizuoka University in 1998. He received his PhD under the supervision of Professor Akira Isogai at the Intercellular Communications Laboratory at Nara Institute of Science and Technology (NAIST) in 2004. Subsequently, he worked as a Japan Society for the Promotion of Science (JSPS) Postdoctoral Fellow for Research Abroad in the Tai-ping Sun laboratory at Duke University in the U.S.A. from 2005 to 2007. He joined the Toshio Hakoshima laboratory at NAIST as a GCOE Research Fellow and JSPS Research Fellow from 2007 to 2011. He returned to the Intercellular Communications Laboratory (Professor Seiji Takayama) at NAIST as an Assistant Professor in 2011. Later, he moved to the Bioorganic Chemistry Laboratory at the University of Tokyo as a Project Associate Professor in 2017. His major research themes are the molecular and structural biology of self-incompatibility in plants and the plant hormone gibberellin. He has determined the structures of ligand-receptor complexes of gibberellin and self-incompatibility determinants. He received the Japan Prize in Agricultural Sciences, Achievement Award for Young Scientists in 2014.

Seiji Takayama was born in Tokyo, Japan, in 1959 and graduated from the University of Tokyo in 1981. He received his PhD in 1986 from the University of Tokyo under the supervision of Professor Akinori Suzuki. He then joined Ajinomoto Co., Ltd. where he was engaged in drug seed discovery until 1995. During that time, he joined the University of California, San Diego School of Medicine in the U.S.A. as a postdoctoral fellow from 1991 to 1993. In 1995, he became an Associate Professor of the Intercellular Communications Laboratory (Professor Akira Isogai) at Nara Institute of Science and Technology (NAIST), and in 2006, he was promoted to professor at NAIST. Since 2016, he has been a professor at the Graduate School of Agricultural and Life Sciences at the University of Tokyo. His main research interest is plant self-incompatibility, which he began in 1983 as a PhD student under the direct supervision of then Associate Prof. Akira Isogai. Currently, he is working to elucidate the mechanisms by which plants acquire diversity, including research on plant interspecies incompatibility. He was awarded the Japan Prize of Agricultural Science in 2013.

Akira Isogai was born in Tokyo, Japan, in 1942 and graduated from the Faculty of Agriculture, the University of Tokyo in 1964. After working in a private company 6 years he was appointed as an Assistant Professor in the laboratory of Professor Saburo Tamura at the Faculty of Agriculture, the University of Tokyo in 1970. He received his Doctoral Degree of Agriculture in 1973. He was promoted to Associate Professor in the Faculty of Agriculture, the University of Tokyo in 1980. He then moved to the newly established Nara Institute of Science and Technology (NAIST) as Professor of the Laboratory of Intracellular Communications in the Graduate School of Biological Sciences in 1994. He was elected as President of NAIST in 2009 and served a 4-year term. His research field is bio-organic chemistry, which deals with bioactive compounds related with various biological phenomena in plants, microbes, and insects mainly based on chemistry. He received the Japan Bioscience, Biotechnology, and Agrochemistry Society Award in 1996. He was awarded the Japan Academy Prize jointly with Kokichi Hinata of Tohoku University to their work on self-incompatibility in plants in 2002 and selected to be a member of the Japan Academy in 2022.