Pollen–Pistil Interaction During Distant Hybridization in Plants

Abstract

A combination of high potential productivity and ecological stability is essential for current cultivars, which is achievable by breeding. Interspecific/intergeneric hybridization remains a key approach to producing new high-yielding and resistant cultivars. Interspecific reproductive barriers (IRBs) appear in the interaction between the pollen and pistil of interspecific/intergeneric hybrids. The mechanisms underlying these hybridization barriers are to a considerable degree unknown. The pollen–pistil interaction is decisive because the pollen of distantly related plant species either is not recognized by stigma cells or is recognized as foreign, preventing pollen tube (PT) germination and/or penetration into the stigma/style/ovary. This review mainly focuses on (1) the pollen–pistil system; (2) IRB classification; (3) similarity and differences in the function of self-incompatibility (SI) barriers and IRBs; and (4) physiological and biochemical control of IRBs and their overcoming. The main goal is to illuminate the physiological, biochemical, and molecular mechanisms underlying the growth arrest of incompatible PTs and their death. In general, this review consolidates the current understanding of the interaction of the male gametophyte with the sporophyte tissues of the pistil and outlines future research directions in the area of plant reproductive biology.

1. Introduction

The transition to high-yield and ecologically friendly agriculture implies the expansion in diversity of cultivars and hybrids of the main agricultural crops as well as the boost in crop yields, quality upgrade of agricultural production, and increase in the plant resistance to biotic and abiotic stresses [1,2]. Currently, traditional breeding approaches fail ever more frequently in attaining the target results because of the limited genetic resources of the plants involved in traditional breeding [3]. In the majority of cases, only the plants of the same species or very closely related species are able to successfully cross [4]. However, representatives of other genera in the same families as the crop plants frequently carry valuable agronomic traits (first and foremost, resistance to pathogens, tolerance of abiotic factors, growth behavior, fruit quality, and ornamental features) of prime interest to breeding [5]. In the absolute majority of cases, the involvement of plants of different genera within the same family in breeding is unfeasible because of a number of interspecific reproductive barriers (IRBs) [6,7]. The stigmata either fail to recognize the pollen of distantly related plant species or recognize it as foreign, thereby interfering with the processes of fertilization, embryo development, and seed setting [8,9]. Distant hybridization comprises the crosses between two different species, genera, and higher rank taxa [10], and overcoming reproductive barriers in this case is the key to success.

Although there has been a constant interest of researchers in this problem (as is suggested by several reviews [9,11,12]), our current understanding of the pollen interaction with pistil in the IRBs is very limited, and the underlying physiological and molecular mechanisms remain, to a considerable degree, vague. Some earlier studies focused on the insight into the reproductive barriers before fertilization, which prevent a successful zygote formation via mechanisms such as the adaptation of pollinators or the incompatibility of pollen and pistil [13,14]. According to the recent advances in the area of molecular biology, the postzygotic mechanisms are regarded as significant drivers of plant speciation [15]. These mechanisms act at different moments in time, thereby leading to a decrease in the viability of hybrid seeds or seedlings or to the sterility of resulting hybrids [11]. The incompatibility of reproductive elements during pollination and fertilization in plants appears as the inability of pollen tubes (PTs) to pass the overall length of the style and/or to implement fertilization, although both the style and pollen are completely active in terms of their function [16]. Reproductive incompatibility is observable in both interspecific and intraspecific crosses as well as in self-pollination [11]. In this review, we focus on the physiological, biochemical, molecular, and some other aspects of the PT growth arrest in distant hybridization.

Scientists attempt to utilize different methods to overcome the incompatibility in distant hybridization, such as chromosome duplication, bridge species, protoplast fusion, and embryo rescue [7]. Last year was marked by the 125th anniversary of Grigory Dmitrievich Karpechenko, a prominent Russian scientist, who was the first to create the fertile distant hybrid between the cabbage and radish (Raphanobrassica) [17]. This started many further scientific research studies into distant plant hybridization.

Note that agricultural biotechnology has allowed researchers to introduce novel properties into the main widespread crops that increase their yields; however, the endless debate on the benefits and hazards of genetically modified plants goes on, and the relevance of traditional breeding methods has not decreased.

2. Pollen–Pistil System

Similar to all plant systems, the reproductive traits are the final result of molecular interaction and the genetic mechanisms that generate diversity [9]. The pistil of angiosperm plants is a unique system (Figure 1). It plays a protective role and acts as a vehicle for PTs, allowing them to grow up to the ovary; provides the site where a pollen grain (PG) can interact with the pistil; and regulates the growth of PTs and, correspondingly, fertilization [18,19]. The surface of the stigma is the receptor site for triggering the mechanisms associated with pollination and fertilization, as well as the initial point for determining incompatibility [20].

The interaction of pollen and pistil switches on the adhesion of PTs; their hydration; germination; penetration into the stigma; ingrowth into the style; further growth inside the specialized hollow channel as, for example, in lily (Figure 2a) or along the pistil conductive tissue as, for example, in petunia (Figure 2b–e); penetration into the embryo sac; and the possibility for further gamete fusion and embryo development [20,21]. This chain of events reflects the interaction of the male gametophyte with female sporophyte tissues and with the female gametophyte at the final stage. This particular interaction determines the behavior of the male gametophyte at the stage of its function when it exists in an intimate relationship with the living tissues of the other genotype. IRBs can act at each of the described stages (Figure 2). We focused on the pollen–pistil interaction that enhances the PT selection and interspecific/intergeneric pollen rejection. The goal of our review is to shed light on the physiological, biochemical, and molecular mechanisms underlying the growth arrest of incompatible PTs and their death.

2.1. PG Adhesion

Below, a brief description of the individual stages in the pollen–pollen tube–pistil interaction can be found. Once PGs land on the stigma, they attach to its surface (adhesion) (Figure 2c,f). It is the adhesion that initiates the process of intercellular recognition. This is the first stage in the interaction between the pollen and stigma. The adhesion is mainly determined by the humidity of pollen and stigma. The nature of adhesion has been long studied by different methods, and various compounds displaying adhesive properties—arabinogalactans, monosaccharides, glycoproteins, glycolipids, and some others—have been identified [22]. Four types of pollen adhesion to the stigma have been found when studying Brassica oleracea, namely, (1) primary, which takes place immediately after the pollen contacts papillae (both compatible and incompatible); (2) secondary, observable only 15 min after the contact and continuing for approximately 90 min until the PT emergence (observable only on compatible stigmata); (3) the contact of PT with the cuticle (only on incompatible stigma); and (4) the contact between growing PT and papillae surface [23].

Pollen adhesion serves as a precondition for pollen hydration, subsequently enabling pollen germination and tube penetration [24]. Extensive research on this process has been conducted in the Brassicaceae family. Members of this family possess stigmas covered with a waxy cuticle and a proteinaceous pellicle, characterized by the absence of secretory fluid during pollination (Figure 2f).

Measurements of pollen adhesion forces revealed that cross-pollination between Brassica and Arabidopsis resulted in poor pollen adhesion [25], indicating pollen selectivity at this stage. Pollen adhesion in Brassicaceae is believed to involve two distinct phases. Pollen capture is likely mediated by biophysical or chemical interactions between the stigma surface and pollen exine [24,26].

The pollen wall’s complex structure—shaped by exine deposition and assembly—varies markedly across species. These structural differences likely play a role in both interspecific and intraspecific pollen recognition [27]. Experiments with exine-stripped Arabidopsis pollen fragments revealed their remarkably strong adhesion to stigma surfaces—exceeding that of pollen from other plant families. This demonstrates a clear species-specific bias in pollen capture [25].

Conversely, research also showed that removing the waxy cuticle of papillary cells in Brassica oleracea completely suppresses their ability to capture pollen grains [28]. The pollen exine primarily consists of sporopollenin, whose deposition and polymerization are critical factors in determining exine wall patterning. Sporopollenin, composed mainly of aliphatic and phenylpropanoid derivatives, exhibits significant diversity across taxa [29].

2.2. PG Hydration

Most pollen grains become highly dehydrated upon release from mature anthers, which enhances their dispersal capacity and survival [24,26,30]. Consequently, these desiccated pollen grains must rapidly acquire water, nutrients, and small molecules to achieve: (1) metabolic reactivation, and (2) exine wall breakdown through combined hydrolytic activity and turgor pressure—processes that ultimately drive pollen germination and tube growth [31].

Pollen is reactivated on the stigma surface during its hydration, which is mainly provided by the water passing from the stigma tissues. Successful fertilization requires that the pollen and the pistil are distinctly coadapted to one another. However, the water flow regulated by physicochemical factors is not the only component of the process that controls the pollen hydration on the stigma surface. The exudate of the species with wet stigmata contains large amounts of water and serves as a favorable medium for PG germination (Figure 1a,c). As has been shown, the lipid component of the exudate plays an important role in hydration in the species with both wet and dry stigmata, for example, Arabidopsis [32]. The receptive surface of the dry stigma is covered by a protein pellicle, containing lipid components, to which pollen attaches [33]. Some data suggest that the hydration rate is a player in certain types of self-incompatibility (SI) [34]. In particular, the SI of Brassicaceae plants is of a sporophyte type, the incompatible pollen is rejected directly on the stigma, and the hydration rates in compatible and incompatible pollinations are different (Figure 3a,b) [35].

In Brassicaceae, the release of hydration-inducing molecules from the stigma depends on pollen–stigma recognition. During the initial hydration phase, the pollen foot serves as a medium where pollen coat lipids and proteins mix with molecules from papillary cells, enabling signal exchange. Reactive oxygen species (ROS) play a crucial role in this process.

Before pollination, RAPID ALKALINIZATION FACTOR 23/33 (RALF 23/33) triggers ROS production in papillary cells via the ANJEA–FERONIA–ROP2–respiratory burst oxidase homolog D (ANJ-FER-ROP2-RBOHD) pathway. During compatible pollination (Figure 3a), POLLEN COAT PROTEIN B-class (PCP-B) from the pollen coat binds to the LORELEI (LRE)-LIKE GLYCOSYLPHOSPHATIDYLINOSITOL (GPI) ANCHORED PROTEIN 1/ANJEA/FERONIA (LLG1/ANJ/FER) complex, suppressing RBOHD-dependent ROS and reducing intracellular ROS levels, thereby allowing water transport for pollen hydration [36,37].

The molecular network of stigma-derived and pollen-carried factors regulates pollen selectivity for compatible hydration while acting as an effective barrier against incompatible pollination [30] (Figure 3b). Subsequently, the papillary cell near the pollen attachment site releases compatibility factors via polarized secretion. The released fluid is absorbed by dehydrated pollen grains through an efficient channel formed by the restructuring of pollen coat lipids, facilitating metabolic reactivation of pollen grains [38,39]. SC pollen enhances ethylene signaling, induces PCD in stigma papilla cells, and leads to pollen growth and penetration into the stigma [40,41] (Figure 3a). In case of self-incompatible pollination, papillary cells do not die off (Figure 3b).

2.3. PG Germination and PT Growth

After the hydration, pollen germinates starting from the phase of imbibition, which is commonly followed by the lag phase, PT emergence, and elongation. This process comprises an increase in respiration intensity, activation of mitochondria, utilization of pollen reserve substances, and development of Golgi bodies, involved in the growth of plasma membrane and formation of PT wall [18,21]. PTs grow further in the stigma and style tissues (Figure 2d). Individual PT regions or zones are functionally specialized. Typically, four zones are distinguished, namely, (1) apical and (2) subapical; characteristic of both are abundant elements of the Golgi apparatus, vesicles in the cytoplasm, and fibrillar wall; (3) nuclear zone, harboring the vegetative nucleus and generative cell, surrounded by two membranes (the inner layer of the wall surrounding this zone contains callose); and (4) the zone of vacuolation and formation of the callose plug (Figure 4a).

Characteristic cytological abnormalities determined by the PT incompatibility with the stigma, style, or ovary tissues are evident when studying the PT apex [42,43]. The substances of the stigma and style are also very important: disturbances of nutrition regulation are observable in incompatible pollinations. Another of the most important consequences of pollination is the redistribution of metabolites.

As noted above, open (hollow) styles are distinguished (Figure 2a); it is assumed that this style type evolved as a result of incomplete fusion of the conduplicate stylode (family Liliaceae) [21]. The canal of the open column is filled with fluid secreted by the tubular cells lining it. It is in this fluid that PTs grow. Closed (solid) styles (Figure 1 and Figure 2b) feature one or more strands of conductive tissue in the central part of the style, either immersed in the main tissue of the style or connected with vascular bundles. In the mucilaginous apoplast of the conductive tissue, PTs grow [20]. However, in some representatives of angiosperms (for example, the family Poaceae), the conductive tissue lacks an extensive intercellular matrix, and its cells are tightly packed. In such styles, the PTs grow by pushing the cells apart. Semi-closed styles are also distinguished (Fabaceae) [44].

Numerous reviews [18,21,45] show a constant interest of researchers in PTs. The emergence of PTs was a fundamental advance in the evolution of terrestrial plants [46]. Actually, PTs allowed the plants to get free from the need for water during reproduction. The importance of this simple organism, the male gametophyte, is also determined by its specific cytological structure, with its characteristic apical growth and the cell wall lacking a canonical structure but perfectly adapted to the growth mechanism [47]. Concurrently being the decisive factor of sexual reproduction and a perfect cellular model, PTs have become most attractive to researchers worldwide. In addition to its fascinating biology, the PT represents an excellent experimental system. Unlike most plant cells that dedifferentiate and lose polarity during in vitro cultivation, cultured pollen maintains its polarity and developmental identity. In vitro, pollen tubes grow synchronously and uniformly, demonstrating highly polarized cytoplasmic organization with an apical region filled with exocytic vesicles (Figure 4, bottom left quadrant).

PT growth is an intricate process, summating the main mechanisms of cell expansion: secretory vesicles transport polysaccharides and enzymes to a certain site, and the set of molecules and proteins translate the external signals, which influence the cytoskeleton of PTs and, consequently, their structure and growth direction (Figure 4a) [48]. This process is also controlled by regulatory mechanisms intended to provide the growth of “foreign” PTs and the removal of “own” PTs, which is the phenomenon referred to as self-incompatibility (SI) [49]. This is an efficient barrier to reproduction and the mechanism promoting the crosses of genetically different plants but is concurrently a limiting factor in the case of the plants of agricultural importance [11]. Thus, plants have had the chance to enhance genetic variation, which is an important basis for evolution [50,51].

Molecular signals such as transmitting tissue-specific (TTS) in tobacco (Nicotiana tabacum) [52] or gamma-aminobutyric acid (GABA) in Arabidopsis [53] are localized in pistil tissues and guide the PTs to deliver sperm cells to the embryo sac for fertilization. Moreover, female tissues provide attraction signals for PTs, as demonstrated using mutants with delayed embryo sac development that fail to attract emerging PTs [54]. Over ten years ago, two principal biochemical mechanisms governing PT elongation were discovered: a steep calcium gradient at the PT tip and the contribution of actin microfilaments to the elongation process (Figure 4a).

A key role in PT growth is also attributed to pectin methylesterases (PMEs), which are essential for cell wall formation and require strict regulation of enzymatic activity, likely achieved through environmental properties, with pH being one of the key parameters. In particular, local pH reduction due to de-esterification may promote cell wall relaxation by stimulating the activity of several hydrolases that weaken the cell wall, such as polygalacturonases and pectate lyases [55]. The interplay between these opposing effects—cell wall stiffening by PME and calcium crosslinking versus cell wall loosening by various hydrolases—likely regulates the directional growth of the pollen tube tip.

In their review, Zheng et al. [48] summed up the distinct organization of actin in PTs, focusing on the short but dynamic actin filaments in the apex and the proteins capable of regulating their dynamics. The role of Rops/Racs in pollen development is well established, and for a detailed description, the reader is referred to recent reviews [56,57]. Briefly, in Arabidopsis, AtRop1, AtRop3, AtRop5, AtRop9–AtRop11, and AtRop8 are expressed in pollen, and some of them have been implicated in regulating tip actin dynamics and possibly calcium ion gradients. For example, AtRop1 has been shown to regulate pollen germination and tube growth [58].

Insight into the role of the actin cytoskeleton is important for understanding numerous events in the PT, including the SI process and IRBs. As soon as the PG reaches the receptive stigma and PT commences its growth, it has to undergo a long journey in order to transport two sperm cells to the embryo sac. The long way of PT through the stigma and style to the seed aperture is the key topic of the review by [59], where the authors emphasized the precise regulation and molecular dialogue necessary for fertilization. In addition to the basic research, the insight into this interaction is important for the control of the fertilization process.

2.4. PT Ingrowth into the Ovary and Gamete Fusion

The incompatibility can take place when PT enters the ovule; fertilized egg cells or young embryos are aborted as a result of incompatibility after karyogamy [59]. As is earlier described (see Section 2.2), PTs are hydrated and germinate on the stigma surface; the PTs grow via polarized elongation through the conductive tract of the pistil tissue and carry two sperm cells over the entire way to the embryo sac in the ovary. The female gametes (egg cells) and central cells develop in embryo sacs, located in the ovule. In addition to the female gametes, embryo sacs also contain synergids and distal antipodes (Figure 2b,f (right inserts)). A directional growth of the PT apex to the female gametophyte depends on the chemoattractants secreted by synergid cells [60,61]. A failure at any stage in the pollen–pistil interaction leads to infertility and, consequently, may be part of the mechanism that either actively blocks the interspecific pollen or passively disfavors the interspecific pollen as compared with the pollen of the same species [11,62].

The endosperm is necessary as a nutritive tissue for the growth and support of the embryo. The development of endosperm is decisive for the development of the embryo and the production of a viable and healthy progeny. In a general sense, the disturbance of endosperm development can be a cause of the lethality of hybrid seeds, referred to as hybridization barrier based on the endosperm in different groups of species with a nuclear endosperm, such as Arabidopsis [63], rice [60], and Capsella [64], as well as in the species with a cellular endosperm, such as Mimulus [65] and tomato [66] (Figure 2e).

3. Classification of IRBs

Abundant histological data on pistillate flowers have revealed at least four variants of the site where PT growth is arrested, which reflects the unique mechanism providing the recognition/selection of male gametophytes [16] (Figure 2). According to the classification by Sears [67], the incompatibility response manifests itself at different stages of pollination and fertilization: (1) the inhibition takes place before or during pollen germination, that is, on the outer surface of the stigma; (2) the growth of PT is slowed down during its advance along the style conductive tissues; (3) when PT enters the ovule; and (4) fertilized egg cells or young embryos are aborted after karyogamy as a consequence of the incompatibility response. The current classification is much more complex (Figure 5).

In the relevant literature, the terminology associated with different IRB types is considerably diverse (Figure 5). The totality of IRBs fall into bilateral and unilateral variants; the former prevent the emergence of successful hybrids independently of the particular species acting as a female or a male parent and the latter, allow for successful hybridization when a certain species acts as a female (or a male) parent but reciprocal crossing direction never produces successful hybrids [16,68].

Different IRB types are also divided depending on the time period when they act in the reproductive sphere. In a broad sense, two main IRB types are distinguished, namely premating and postmating (before and after pollination).

3.1. Premating IRBs

The premating IRBs comprise spatial, temporal, and mechanical barriers, as well as the morphological specialization influencing the behavior of pollinators [16,69] (Figure 5). Geographic isolation, flowering phenology, and ecological factors, such as soil type and preferences of pollinators, can influence IRBs before the pollination. The IRBs controlled by pollinators are abundant among the Solanaceae, and the genes that control the flowering characteristics, such as petal color, shape of the corolla, nectar production, and secretion of volatile aroma molecules, can serve as speciation genes [70]. In particular, Hoballah et al. [71] have identified a transcription factor that controls the color of petunia petals and demonstrated that the sequence variation in the AN2 (ANTHOCYANIN2) gene generates different pollination syndromes that change the preferences of pollinators and thereby isolate populations.

A complete fidelity to pollinators is rather rarely met; correspondingly, speciation as a rule is determined by pollinators and does not lead to an absolute reproductive isolation [72]. Dell’olivo et al. [73] studied the sympatric populations of Petunia axillaris and P. integrifolia in Uruguay and discovered that a strong but not absolute premating IRB generates the preference of pollinators. They measured the strength of all potential IRBs between two species and reported that the IRBs after mating but before fertilization were almost as strong as the isolation mediated by pollinators [73].

3.2. Postmating IRBs

3.2.1. Prezygotic Postmating IRBs

Two types of postmating IRBs are distinguished, namely, prezygotic and postzygotic (Figure 5). The prezygotic postmating IRBs comprise one or several of the numerous stages in the pollen and pistil interaction, which commence from the landing of pollen on the stigma surface, followed by the progamic stage of fertilization, and end with the fertilization of egg cells, that is, all stages described by Sears [67]. The disturbed pollen germination, poor pollen penetration through the stigma, weak PT growth, and pollen retention in the gynoecium are the prezygotic barriers (Figure 2c,d,f). Some researchers believe that the SI mechanisms enhancing these barriers include epigenetic silencing [74], which implies the potential role of epigenetics in the prezygotic barriers. However, only a few pieces of evidence link epigenetics and prezygotic barriers.

3.2.2. Postzygotic Postmating IRBs

The postzygotic barriers act after fertilization has taken place and can result from inviability of the zygote, underdevelopment of the embryo or endosperm, and inability of the hybrid seedling to develop; an inadequate growth of the endosperm leading to the abortion of the embryo is also observable, which results from insufficient nutrition, hybrid sterility, and lethality caused by chromosome or genetic variations [37] (Figure 5). The postzygotic postmating barriers are also divided into two types, namely, extrinsic and intrinsic [75]. The extrinsic barriers imply an interaction with the environment; for example, the hybrids display reduced growth in the parental habitats, even in the background of typical development. The intrinsic barriers manifest themselves independently of the environment, for example, as hybrid inviability or sterility [76].

3.2.3. Incongruity

Many researchers introduce the concept of incongruity, regarding it as a passive process versus incompatibility, regarded as an active rejection [16]. Lee et al. [77] emphasized that the Nicotiana species with long pistils, as a rule, display a higher PT growth rate as compared with the species with short pistils; correspondingly, the pistil length is a potential reproductive barrier in the crosses of the species with long pistils and with short pistils. Solanum pennellii has one of the longest pistils in the genus [77]. The need for HD-AGP (histidine domain–arabinogalactan proteins) congruity in the pollen–pistil interaction can also explain the conspecific pollen precedence, the phenomenon in which the conspecific pollen is more effective in seed setting as compared with heterospecific pollen when they compete within the same pistil [16]. In nature, the pollinator preference in combination with conspecific pollen preference can be sufficient for the isolation of S. lycopersicum and S. pimpinellifolium from the rapidly growing S. pennellii pollen [62].

Evidently, incompatibility is a more useful paradigm for pollen rejection. A theory suggests that the prezygotic IRBs are associated with SI. This theory relies on the components of the outbreeding system based on SI for an active inhibition of heterospecific pollen on the stigma or in the pistil. The incongruity covers all other prezygotic IRBs, suggesting that it is based on the passive mechanisms manifesting themselves in a nonoptimal pollen function in a heterospecific pistil [16]. The discrepancy includes the priority of the pollen of one species when the PTs of this species display better growth in the pistil tissues. IRBs play a key role in the evolution of species because they strengthen the boundaries by decreasing or blocking the hybridization between nascent species [78].

4. Similarities and Differences in the Function of SI Barriers and IRBs. Unilateral Incompatibility (UI)

Currently, the SI mechanisms are the best studied in the pollen–pistil interaction on molecular and genetic levels, allowing them to be used as the model for the pollen rejection processes in interspecific pollination as well [11]. As is believed, SI emerged approximately 90 million years ago as the evolutionary mechanism preventing self-pollination and providing genetic diversity [79]. SI has many times independently evolved in different plant groups and is currently observed in many families [36]. SI prevents self-pollination in over 100 families and approximately 40% of plant species [80]. This mechanism enhances the successful spread and the diversity of flowering plants, which is important for adaptation. At the moment, the reviews on this topic are sufficient, and a comprehensive discussion of this problem is beyond the scope of our review [61,81]. In Figure 3 and Figure 4, we present highly simplified schematics of sporophytic (Brassicaceae) (Figure 3) and gametophytic (Solanaceae) (Figure 4) SI mechanisms for easier comprehension of aspects covered in our article.

In plants, the S locus is involved in the control of SI. The structure of this locus, the nature of the genes it codes for, and the mechanisms underlying SI in different plant families are unique [82]. The SI can be heteromorphic and homomorphic (Figure 5). Heteromorphic SI is observable in the population, with the flowers differing in their morphology. As early as 1877, Darwin [83] described the existence of two or three flower types within the same species, referring to them as morphs. Flower morphs differ in the lengths of the style and anther filaments. An example is Primula vulgaris with its alternating long- and short-style flowers and the outcrossing reached via structural barriers rather than the interaction between pollen and pistil [83].

Homomorphic SI is observed in species with identical flowers. In this case, the S locus codes for the genes expressed in the pistil and provides the recognition and rejection of the self-pollen or the preference for the cross-pollen. Homomorphic SI is subdivided into gametophytic and sporophytic types (Figure 5). In the gametophytic SI, pollen compatibility is determined by its own S haplotype. Pollen does germinate, but the PT growth is arrested after a certain time interval. This type is characteristic of the family Solanaceae (petunia, tobacco) (Figure 4b). The response of pollen in a sporophytic SI is determined by the genotype of the diploid maternal plant and the interaction between the alleles of SI genes present in the pollen and pistil. In this case, the germination of PGs is inhibited on the stigma (Figure 3b). This type is characteristic of the family Brassicaceae. In any case, the S locus codes for the genes that control specificity on the part of both the pollen and pistil. So far, the genes of three different SI systems have been studied in detail, as well as their functions [81,84]. Figure 5 shows the generalized detailed classification of SI.

The search for the proofs of a universal incompatibility mechanism is a promising direction since it is most unlikely that every existing species has developed a unique mechanism. Consequently, it is reasonable to expect that incompatibility is provided by a universal mechanism. On the other hand, researchers [62,85] are most likely right in that the unifying theory will be based on a polygenic rather than a monogenic control of the PT growth inhibition.

Some authors [62,86] consider the interspecific incompatibility in a tight connection with SI, relying on the analogy of the PT growth arrest in distant hybridization to SI. The hypothesis of a universal incompatibility mechanism is based on the existence of specialized, developed fertilization responses that emerged in the interaction of similar alleles controlled by the S locus. However, different unrelated genes interact in the case of interspecific incompatibility because the chance for similar alleles to meet during distant hybridization is very small. Nonetheless, both mechanisms—SI and IRB—are directed to prevent the undesirable fertilization: SI is intended for self-pollination and inbreeding and IRBs for the prevention of hybridization between different species. The schemes in Figure 3 and Figure 4 summarize the functional parallels and differences in the functioning of reproductive barriers in SI and IRB.

It is generally accepted that interspecific incompatibility is a form of post-pollination prezygotic reproductive barrier, providing the rejection of the pollen of different species in pistil tissues [20,30]. Another type of interspecific incompatibility, frequently occurring among closely related angiosperm species, is unilateral incompatibility (UI), characterized by specific unidirectional pollen receipt by the stigma in reciprocal crosses.

The very assumption that SI plays a role in IRBs is based on the SI × SC (self-compatible) rule, described for different taxa with either gametophytic or sporophytic SI. According to this rule, the SI pistil rejects the pollen of a SC species in the crosses of a self-incompatible female form and a heterospecific self-compatible male form. However, the reciprocal cross (SC × SI) results in successful seed setting. This UI phenomenon suggests the absence of the mechanism of foreign pollen rejection in self-compatible species [87]. In this UI, known as the SI × SC rule [88], the pollen of SI species on the pistils of SC species can lead to the formation of hybrid seeds, but the reciprocal cross (SC pollen on SI pistils) emerges to be ineffective.

Recent studies indicate the correlation between UI and SI in the family Brassicaceae [89]. The SI in this family is controlled by the haplotype of the S locus that comprises two main genes: SRK (S-receptor kinase) and SP11/SCR (S locus cysteine-rich protein). SRK codes for the membrane receptor of serine/threonine kinase, which is expressed in the cells of the stigma surface (Figure 3c). This receptor specifically interacts with the SP11/SCR protein, expressed in pollen. If the pollen and the pistil belong to the same S-haplotype, the corresponding signaling pathway is activated, blocking pollen germination and preventing self-pollination. It is assumed that the heterotrophic pollen of closely related species is rejected in an analogous manner on the stigma via the effect of the SRK protein [20,30,89]. As is shown, the protein SRK receptor in the absence of the interaction with pollen protein SP11/SCR forms the SRK–SRK homodimers, which leads it to its basal state. This prevents an accidental activation of the SI system. However, the interaction with the SLR1 (S locus-related glycoprotein 1), which is not strictly specific to S locus alleles, interferes with the formation of SRK homodimers. This regulates the amount of SRK homodimers, which play a decisive role in the recognition of the self- and cross-pollen, as well as different interspecific pollen types via the SRK-mediated signaling pathway of pollen rejection. Interestingly, UI was observed in an intraspecific cross of B. rapa Turkish and Japanese strains, while UI typically takes place in interspecific crosses [90]. Molecular genetic studies of the intraspecific UI in B. rapa revealed stigma and pollen recognition factors SUI1 and PUI1, respectively. It has emerged that SUI1 and PUI1 are analogous to SI factors, namely SRK for the stigma and SP11 for the pollen, respectively. SUI1 and PUI1 are tightly associated and result from the duplication event of the SRK–SP11 region in Brassica [91]. The physiological phenotype of the pollen rejection in B. rapa intraspecific UI is similar to the SI phenotype [90].

Recent experiments have shown that SRK is able to form homodimers independently of the presence of pollen, which leads to a basal inhibition in Arabidopsis as well [30,89]. In addition, glycoprotein SLR1, associated with the S locus, was shown to be able to interact with SRK and interfere with the formation of SRK homodimers. The levels of SRK homodimers play a decisive role in distinguishing between the self- and cross-pollen, as well as different interspecific pollen types via the SRK-mediated signaling pathway of pollen rejection. Liu et al. [89] have demonstrated that stigma UI3.1, a genetically identified determinant of the UI stigma in the Arabidopsis lyrata × A. arenosa crosses, codes for glycoprotein 1, associated with the S locus (SLR1). Heterological expression of A. lyrata or Capsella grandiflora SLR1 renders some A. thaliana accessions the ability to discriminate the heterospecific pollen. The acquisition of this ability also requires a functional receptor kinase of the S locus (SRK), the ligand-induced dimerization of which activates the pollen self-rejection in the stigma. SLR1 interacts with SRK and prevents the formation of the SRK homodimer. The authors proposed a scheme for pollen discrimination based on the competition between the basal and ligand-induced formations of the SLR1–SRK and SRK–SRK complexes. The resulting levels of SRK homodimer will be accepted by the general pollen rejection pathway, allowing for the discrimination among the conspecific self- and cross-pollen, as well as heterospecific pollen. Their results establish a mechanistic link between the SI and IRB at the pollen recognition stage [89].

Murfett et al. [92] demonstrated that the S-RNase expression of the incompatible species Nicotiana alata in the pistils of the self-compatible N. plumbaginifolia causes a rejection of N. tabacum pollen but not the pollen of its own species (N. plumbaginifolia). However, the N. plumbaginifolia pollen was also rejected when the S-RNase transgene was introduced on another genetic background and expressed in combination with other pistil factors. These results suggest that S-RNase is involved in the rejection of both N. tabacum and N. plumbaginifolia, but the rejection mechanisms differ depending on the genetic background [92]. Further studies discovered a pistil-specific protein, HT, playing an important role in the SI system of Solanaceae plants, such as Nicotiana and Solanum (Figure 4c). According to Hancock et al. [93], HT is a key modifier necessary for the rejection of N. plumbaginifolia pollen. The absence of this protein leads to a disturbance of the pollen rejection mechanism by enhancing PT ingrowth into the pistil tissues. The SC pollen of N. alata was rejected only in the presence of this protein. HT stabilizes the S-RNases responsible for RNA degradation in the inappropriate PTs and boosts their activity. In addition to S-RNase, the HT interacts with the 120 K (120-kDa glycoprotein) proteins to strengthen the barrier functions of the pistil during the recognition of the pollen with SLF (S locus F-box) to regulate the growth of compatible and incompatible PTs. Thus, the S-RNase–dependent mechanisms of interspecific pollen rejection depend on the pollen specificity and the modifier genes providing their coordination [93]. The S-RNase and HT expression in S. lycopersicum, an SC species, induced the pollen rejection for the red-fruited SC tomato species but not for the green-fruited tomato species [94]. Together, these studies demonstrate that the pistil-specific SI and incompatibility barriers have common genetic factors.

The prevalence of the SI × SC rule implies that the use of the SI system to block the gene flow between diverging taxa is most likely the fastest way to this target. The mechanism underlying the IRBs associated with SI is rather vague but most likely includes a locus capable of rapidly mutating and responsible for the inhibition of the heterospecific pollen of all S haplotypes concurrently supporting the intraspecific SI system on a regular basis. However, the IRBs associated with SI do not represent the variant for a compatible strain. Note that SI and UI considerably differ in the level of specificity. SI is exclusively specific and causes only the rejection of the pollen with specific S haplotypes, while the UI associated rejection covers the pollen of the entire species and even genera [92,94]. Lewis and Crowe [95] stated that the pistils with any functional capabilities of the S locus were able to inhibit interspecific pollen of self-compatible strains and, correspondingly, referred to this as a unitary action. The studies of the last decade confirming this assumption [94,96,97] emphasize a considerable difference in the specificity. A precise role played by S-RNase in the arrest of PT growth may differ from its role in the context of SI. For example, it is possible that S-RNases are not involved in pollen recognition but act at later stages, such as in the signaling or inhibiting components of the pollen rejection mechanism. A rapid rejection of the interspecific PTs can be associated with a rapid activation of S-RNase, toxicity connected with an alternative recognition system, or a combination of S-RNase compartmentalization and degradation, which can have different effects on pollen stability in the context of SI and interspecific interactions. The functional studies confirm that the rejection of interspecific pollen is a more intricate process as compared with SI because different factors potentially promote different compatibility and incompatibility mechanisms with the additional factor of excess redundancy [96,97].

Moreover, functional studies confirm that IRBs are more complex than SI and UI in that several mechanisms are involved in these processes. Although SI and UI partially coincide, they are not identical. In particular, there are many exceptions from the SI × SC rule, for example, the SC × SC incompatibility [62], and SI and UI differ in physiological and morphological characteristics. In particular, the interspecific rejection in the stained PTs of pollinated pistils is frequently observed closer to the stigma as compared with the pollen rejection in SI [95,98,99]. In addition, ultrastructural comparison of the SI and UI in S. peruvianum during self-pollination and cross-pollination with S. lycopersicum demonstrates that the PT cell wall thickens during SI rejection but degrades during UI rejection [82,88].

Chalivendra et al. [100] proposed a model system for analyzing molecular mechanisms involved in reproductive barriers. In the wild tomato species Solanum pennellii, both SI and SC populations exhibit UI when crossed with the domesticated tomato. Their study determined the timing of reproductive barrier establishment during pistil development in SI and SC S. pennellii using a semi-in vivo system to track pollen tube growth in developing styles. Both SI and UI barriers were absent in styles 5 days before flower opening but were established 2 days before anthesis, with partial barriers detected during the transitional period 3–4 days before flower opening. The dynamics of known SI factors, S-RNases, and HT proteins were also examined. HT-A protein accumulation showed spatiotemporal correlation with UI barriers in developing pistils. Proteomic analysis of stigmas/styles at key developmental stages revealed a shift in protein profiles from cell division-related proteins in immature stigmas/styles to a set of proteins in mature stigmas/styles that included S-RNases, HT-A protein, and proteins associated with cell wall loosening and defense responses, potentially involved in pollen–pistil interactions.

Subsequently, Tovar-Mendez et al. [94] proposed and tested the hypothesis that S-RNase-dependent UI is sufficient to form interspecific reproductive barriers between red- and green-fruited species by reintroducing functional pistil-side SI genes S-RNase, HT-A, and HT-B into S. lycopersicum. They found that restoring these factors was sufficient to recreate pistil-side interspecific UI barriers, requiring both S-RNase and HT genes. While SI and UI are clearly linked in some cases, the authors demonstrated that this may not hold true for all interspecific reproductive barriers. Notably, HT genes are associated with many UI systems, suggesting their involvement in both S-RNase-dependent and S-RNase-independent pollen rejection mechanisms [94].

Muñoz-Sanz et al. [101] identified a cysteine-rich protein Defective in Induced Resistance 1-like (SpDIR1L) involved in S-RNase-independent pollen rejection in tomatoes (Solanum sect. Lycopersicon), which demonstrates multiple interspecific reproductive barriers (IRB). Some IRBs follow the SI × SC rule, but SC × SC UI rules also exist, enabling identification of IRB factors independent of S-RNase. For example, SC Solanum pennellii LA0716 pistils permit SC Solanum lycopersicum pollen tubes to penetrate only the upper third of the pistil, while S. pennellii pollen reaches S. lycopersicum ovaries. The authors identified candidate genes for the S. pennellii LA0716 pistil barrier based on expression profiles and published results. CRISPR/Cas9 mutants were generated for eight candidate genes and assessed for changes in S. lycopersicum pollen tube growth. Mutants in the gene designated Defective in Induced Resistance 1-like (SpDIR1L), encoding a small cysteine-rich protein, allowed S. lycopersicum pollen tubes to grow into the lower third of the style. SpDIR1L protein accumulation was shown to correlate with IRB strength, and species with weak or absent IRBs against S. lycopersicum pollen share a 150 bp deletion in the SpDIR1L upstream region. These results suggest SpDIR1L contributes to S-RNase-independent IRB.

Another exception from the SI × SC rule consists in the following: instead of always acting in the same direction, the genes responsible for this phenomenon most likely switch on the genes that maintain PG germination and normal PT growth and targeting to the PT ovules growing in compatible pistils. For example, gene PELPIII, coding for an extensin-like protein III. Noyszewski et al. [102] report that the N. tabacum PELPIII influences the PT growth, enhancing fertilization or PT rejection. The inhibition of PT growth was lost when the N. tabacum strain with switched off PELPIII was pollinated with N. obtusifolia and N. repanda [96]. However, no changes in the PT growth were observable between the plants with the switched-off gene and normal plants when the N. tabacum strain with the switched-off gene was pollinated with the N. tabacum, N. rustica, or N. maritime pollen [100]. The TTS (transmitting tissue-specific) gene, coding for the TTS proteins specific to the pistil’s transmitting tract, is also of special interest because these proteins foster the orientation and stimulation of PT growth by interacting with PELPIII proteins. Noyszewski et al. [102] discovered the gene NtPRP (proline-rich protein) in N. tabacum, which is similar in its structure to TTS. A similar protein carrying a PhPRP1 (proline-rich protein 1) domain was found in the P. hybrida pistil tissues [103]. Further studies discovered and described the proteins of this type in P. axillaris (PaaPRP1), N. alata (NaPRP4), Capsicum annuum (CaPRP1), S. lycopersicum (SlPRP1), and S. tuberosum (StuPRP1) [16].

The white-flowered P. axillaris, pollinated by moths, and red-flowered P. exserta, pollinated by birds, are reproductively isolated from both P. integrifolia and P. inflate, which have purple flowers, are pollinated by bees and other day pollinators, and display a strong SI. The pollen of P. axillaris is rejected on the P. integrifolia pistils with the characteristics typical of SI. However, the IRBs in the crosses between these species violate the SI × SC rule because the reciprocal cross is also unsuccessful [16].

The corresponding SI studies of the apple subtribe (Malinae) suggest that the associated genes most likely do not code for SI determinants because the distorted segregation locus is localized to chromosome 5, whereas the S locus is to chromosome 17 [104]. Moreover, the syntheny between these two chromosomes was unobservable. Recent studies of the SI in the sweet cherry (Prunus avium) suggest the potential existence of modifier genes that influence the SI response, differing from the recognition mechanism for distinguishing between self- and cross-pollen [105]. A putative modifier gene, MGST, was discovered in the P. avium SI; a mutation in this gene resulted in the SC of the studied cultivar. MGST can be important for the SI specificity of the genus Prunus. In tomatoes, the self-incompatibility modifiers Cullin1 (CUL1) and HT are involved in the formation of the interspecific barrier [94]. In addition to them, the pollen candidate genes SBP (S-RNase binding protein 1) and SSK1 (Skp1-like protein), associated with the SI response of S-RNase type, were found. The SSK1 and Cullin1 and/or SBP1 proteins form the SCF complex with the SLF (S locus F-box) protein of pollen, which leads to ubiquitination of foreign S-RNases and their subsequent degradation by the 26S proteasome [106]. The genes similar to Skp1 and SBP are located in the distorted segregation locus and are phylogenetically distant from the SI-associated genes. Nonetheless, their high expression in pollen suggests a potential importance for the reproductive process.

Although many studies suggest that the genes associated with SI are responsible for the interspecific barrier, recent data show the existence of other IRBs that are not connected with these genes [107]. The T2 ribonuclease (T2 RIB) of Citrus grandis, involved in the SI mechanism, considerably inhibits PG germination and PT growth. The RIB expression level in SC stigmata is considerably higher compared with the cross-pollinated stigmata. A certain set of candidate genes can be potentially associated with the intergeneric barrier. Many agricultural plant species are polyploids. Zhang et al. [108] have shown that the genetic material of the Triticum aestivum, an allopolyploid (T. turgidum (AABB) × Aegilops tauschii (DD)), consists of several genomes and that expression of the genes of one genome prevails over their homologs in the other genome. This means that the expression level of the corresponding genes is similar to the level of one of their parents and that a high share of T. turgidum genes displays dominant expression in the hybrid. Although the polyploidy in plants has potential advantages in the growth vigor (heterosis), allopolyploidization typically causes a genomic shock resulting from imbalanced and antagonistic gene expression in polyploids [109].

It is well known that the combining ability in many respects depends on the genotype and ploidy level of the parents, which in itself is the point of dispute in the genus Abelmoschus [109]. The use of wild congeners in crop breeding is limited by different pre- and postzygotic barriers. The published data on the pre- and postzygotic barriers in okra crosses are mainly confined to the Abelmoschus esculentus × A. manihot var. tetraphyllus hybridization. The study by Patel et al. [109] is focused on revealing the hybridization barriers between A. esculentus and its wild relatives, A. manihot var. tetraphyllus and A. moschatus, as well as the strategy for overcoming these barriers for a successful introgression of the target traits, allowing for the improvement of okra.

Thus, the relevant literature described the cases when the IRB mechanism is very similar to the SI mechanism. Nonetheless, this issue remains disputable. Certainly, the insight into the SI systems can clarify the control of the pollen flow between species. Currently, SI is the best studied example of the interaction between the pollen and the pistil involving the reproductive barriers mechanism; this is an outbreeding mechanism that prevents fertilization by genetically related pollen. Together, these SI and IRB processes determine the potential of genetic relations for successful pairing: SI prevents pairing between very close individuals of the same species, while IRB pollen rejection prevents too distant crosses. SI evidently promotes the long-term success of a species because the SI strains are genetically diverse and very resistant.

5. Physiological and Biochemical Control of IRBs

Although some molecular mechanisms underlying the regulation of IRBs have been discovered, the data on physiological and biochemical control of these processes are sparse. It is known that signaling pathways influence the pollen recognition by stigma cells. The roles of phytohormones, reactive oxygen species (ROS), and programmed cell death (PCD) in the growth arrest and death of incompatible PTs have not been studied beyond the SI system. Physiological mechanisms, signal reception, recognition of foreign pollen, and the mechanism of PT growth arrest are also vague (Figure 4c). In this chapter, we are focusing on the key advances in our understanding of the molecular, physiological, and biochemical foundations of how these interactions contribute to overcoming the barriers at different pollen and the pistil interfaces on the way of PT growth to enhance fertilization by the desirable partners.

As is mentioned in Chapter 3, the surface of stigma cells, the receptor region for PGs, is the starting point for the determination of compatibility. Thus, the pollen and pistil interaction, comprising adhesion, hydration, pollen germination, PT penetration into the stigma, and PT growth along the style tissues, represents significant barriers to interspecific/intrageneric hybridization in plants (Figure 2). This interaction is of paramount importance because the pollen of distantly related plant species remains unrecognized or is recognized as foreign by stigma cells and is unable to germinate into PT or enter the stigma/style. This stage can be regarded as the recognition stage. Our current understanding of how the pollen and pistil interaction acts as barriers for interspecific/intergeneric hybridization is mostly limited, and the corresponding physiological and molecular mechanisms remain unknown.

Recently, our team extensively studied the SI mechanism in Solanaceae (Figure 4a,b). We succeeded in demonstrating that the hormonal signals and their receptors control intercellular interactions, playing an important role in the PT growth and its regulation during compatible and self-incompatible pollinations. As is shown, the growth of self-incompatible PTs is slowed down in the pistil conductive tissues on the background of intensive ethylene production and elevates the levels of ABA and cytokinins [110]. Three potential targets for hormone action have been detected, namely, H⁺-ATPase of the PT plasmalemma, its actin cytoskeleton, and Ca²⁺-dependent K⁺-channels [111]. ABA and ethylene were shown to be the factors involved in the control of water transport in the germinating petunia male gametophyte [112]. PCD is a factor in the S-RNase type SI in P. hybrida E. Vilm. and self-incompatible wild tomato species [113,114]. The growth of self-incompatible PTs is arrested when the activity level of caspase-like proteases increases during the first hours after pollination, and all PCD signs become evident by the moment of the growth arrest. The treatment of petunia stigmata with caspase-3/DEVDase inhibitor allows the SI to be removed [113]. In this process, fertilization and the setting of seeds took place. Hormonal factors (ethylene and cytokinin) influencing the PCD progress in the incompatible PTs were shown to be involved [115]. Our data demonstrate that ROS are involved in the self-incompatible PT growth and its arrest [116]. These results suggest that the mechanisms underlying the arrest of PT growth and their further death in the case of intergeneric incompatibility are similar; however, any direct evidence is still absent.

Nonetheless, researchers wonder whether there exist interspecific barriers that depend on the physiological control tools analogous to those acting in the SI, although the involved genetic control elements can be different. The events taking place after the pollination in some interspecific crosses look as if they are passive rather than active pollen recognition [117]. In particular, Hogenboom [117] demonstrates that the PTs of the tomato species with a short style grow insufficiently long to reach the ovaries of the species with a long style; the pollen size of the species with a short style is smaller; and both the intraspecific and interspecific pollen diameters tightly correlate with the PT length. Hogenboom [117] assumes that the small pollen of the species with a short style has insufficient resources to develop PTs long enough to reach the ovaries of the species with a long style, which represents an asymmetric mechanical reproductive barrier. The reproductive isolation mechanisms of this type, in combination with additional pollen–pistil incompatibility, are of special importance for the closely related species with common pollinators situated in geographic vicinity [118].

Presumably, the incompatibility barrier acts on a species-specific basis. Different models and observed responses can represent different types of interspecific barriers even within the same family, for example, in Quercus [4,119]: (1) PTs stop at the stigma (Q. coccifera); (2) the absence of PG adhesion, considerably limited PT penetration, and callose deposition in the cells of stigma receptive surface (Q. faginea and Q. robur); and (3) PTs stop at the level of conductive tissue without any further elongation (Q. ilex).

5.1. PG Adhesion in IRBs

The stigmata are classified as wet or dry depending on the presence or absence of secretory fluid during pollination. The plants with wet stigma surfaces, such as tobacco and lily, most likely display a nonselective pollen adhesion, hydration, and germination relying exclusively on the stigma secretory fluids [30] (Figure 1). On the contrary, the plants with dry stigmata presumably discriminate between PGs commencing from the initial stages in pollen adhesion to the PT penetration into the stigma (Figure 2f and Figure 3). In the family Brassicaceae, the Arabidopsis and Brassica species with dry stigmata are able to selectively distinguish between the PGs from the initial adhesion stages to PT penetration [30,120]. In particular, these interactions involve different molecular mechanisms that together regulate the selectivity of compatible pollen or PT and create an effective barrier against an incompatible pollination, thereby promoting the selectivity of species using a method analogous to a multi-key control.

The measurement of pollen adhesion demonstrates that the cross-pollination between Brassica and Arabidopsis causes poor pollen adhesion [121], suggesting a certain level of constraint and species specificity that restricts inappropriate pollen access to adhesion. It is believed that the pollen adhesion comprises two individual stages in Brassicaceae. The initial adhesion, namely, pollen capture, is most likely determined by biophysical or chemical interaction between the stigma surface and pollen exine [24]. It was assumed that a complex structure of the pollen wall resulting from the exine deposition and assembly considerably differs in the external appearance and can influence both the interspecific and intraspecific recognition by the stigma [27].

Thus, the pollen adhesion to the stigma is able to prevent interspecific fertilization at the very early stages of the pollen–pistil interaction based on a weaker adhesion of the interspecific pollen as compared with the pollen of the same species [25].

5.2. PG Hydration in IRBs

As mentioned earlier, in natural environments, the stigma of an open flower is exposed to various pollen types. Consequently, the stigma must discriminate between pollen types for speciation and prevention of undesirable offspring, which is crucial during the early stages of prezygotic reproductive processes.

Plants with wet stigma surfaces, such as petunia and lily, likely exhibit nonselective pollen adhesion, hydration, and germination, relying exclusively on stigma secretory fluids [24,26,30] (Figure 1).

In contrast, as discussed above, plants with dry stigmas presumably discriminate pollen grains from the initial stages of pollen adhesion through tube penetration [30,120] (Figure 2f and Figure 3). Specifically, these interactions involve diverse molecular mechanisms that collectively regulate the selectivity of compatible pollen/tubes while establishing an effective barrier against incompatible pollination, thereby promoting species selectivity in a manner analogous to a multi-lock control system.

In recent years, significant progress has been made in deciphering the mechanisms of selective interactions, providing deeper insights into these discriminatory phenomena. It is difficult to define distinct boundaries between the stages of adhesion and hydration. Recent studies in B. rapa demonstrate that the stigma S-receptor-like kinase (SRK), known for its ability to regulate SI in Brassicaceae, enhances the rejection of intraspecific and interspecific pollen via FERONIA (FER) signal transduction and ROS (Figure 3c). This discovery suggests that this SI system can serve as an interspecific hybridization barrier in plants with SRK. Nonetheless, it is ever more necessary to find out the other types of pollen–pistil interaction that act as the interspecific/intergeneric hybridization barriers in the majority of flowering plants without the sporophytic SI system, as well as in the plants with the gametophytic SI system, such as Solanaceae [121].

In Figure 3, we attempted to summarize the pollen selectivity processes in Brassicaceae, where these mechanisms are currently best understood. Recently, Lan et al. [122] reported that the RAPID ALKALINIZATION FACTOR (pRALF) peptides, expressed in pollen, unblock the stigma barrier resulting from the interaction between Catharanthus roseus RLK1 (CrRLK1L) and the RALF expressed in the stigma (sRALF), necessary for the penetration of pollen. This discovery not only provides a new insight into the interaction between the pollen and pistil but also is important for the production of the hybrids between distantly related plants for breeding purposes [123]. The receptor-like kinases of Arabidopsis FERONIA/CURVY1/ANJEA/HERCULES RECEPTOR KINASE 1 and cell wall proteins LRX3/4/5 interact on the surface of stigma papillae with the RALF1/22/ /33 (sRALF) peptide ligands to form a lock that blocks the penetration of undesirable PTs. The compatible RALF10/11/12/13/25/26/30 (pRALF) peptides received from the pollen promote PT germination, thereby enhancing fertilization. The treatment of the Arabidopsis stigma with pRALF unblocks the stigma barrier, allowing the PTs of distantly related Brassicaceae species to germinate and form interspecific/intergeneric hybrid embryos. Thus, the lock–key system regulating the width of hybridization in Brassicaceae was discovered. This mechanism acts as an interspecific/intergeneric reproductive barrier between the pollen and pistil in the process of PT penetration into the Brassicaceae stigma.

In Arabidopsis, the accumulation of reactive oxygen species (ROS) in the stigma prevents pollen hydration. ROS production is induced by RAPID ALKALINIZATION FACTOR peptides RALF23/33 secreted by the stigma, which are perceived by two stigma receptor-like kinases, ANJEA (ANJ) and FERONIA (FER) of the Catharanthus roseus RLK1L-like (CrRLK1L) family. Interestingly, POLLEN COAT PROTEIN B-class (PCP-B) from the pollen coat caused a reduction in ROS levels in wild-type stigmas after compatible pollination. Finally, it was found that PCP-B and RALF compete for binding to the ANJ-FER complex in a dose-dependent manner, leading to reduced ROS production, thereby promoting hydration of compatible pollen and establishing a gating mechanism for ROS regulation through the downstream effector, the classical Rho-like GTPase-regulated NADPH oxidase pathway RBOHD (Figure 3a) [124].

Nitric oxide (NO) has recently been implicated in compatible pollination. It was observed that NO levels in Arabidopsis stigmas rapidly increase after pollination and then sharply decrease to pre-pollination levels, paralleling the pollen hydration pattern. The rapid NO production depended on the presence of PCP-B in the pollen coat and FER in papilla cells, demonstrating an inverse functional relationship between NO and ROS mediated by PCP-B and FER during compatible pollination. As a result, FER may serve as a target for NO S-nitrosylation, with S-nitrosylated FER showing impaired interaction with its downstream ROP2, thereby disrupting stigma ROS production [20]. Together, these results suggest that the reduction in stigma ROS is a consequence of compatible pollination facilitated by the interaction of PCP-B peptides and the receptor-like kinase FER. PCP-B not only competitively binds FER to interrupt RALF33-mediated ROS production but also triggers rapid NO production to S-nitrosylate FER, thereby inactivating downstream Rac/Rop-regulated RBOH-dependent ROS production.

In Brassica rapa, self-pollination leads to increased ROS levels in stigmas, while decreased ROS levels can disrupt the SI response [20]. Pollen-borne SCR was found to bind the female SI determinant SRK, subsequently recruiting FER and triggering FER-mediated ROS production in SI stigmas, thereby promoting rejection of self-pollen [20]. SI may involve two pathways for SRK-mediated SI responses: one pathway involves degradation of compatibility factors through ARC1, while the other promotes ROS production through FER to prevent pollen hydration. Furthermore, the redox-active properties of ROS raise questions about its potential influence on compatibility factors in the ARC1 degradation pathway.

In cases of distant hybridization, there is significant divergence leading to low affinity between PUI2.1 and SLR1-SRK, which would not promote SLR1-SRK heteromerization, resulting in SRK homomerization maintained at basal levels sufficient to reject heterospecific pollen [125]. Additionally, elevated ROS levels dependent on SRK have been associated with UI pollen rejection in Brassica rapa, similar to the SI response. It was noted that the signal from UI pollen binds to SRK, leading to recruitment of FER and subsequent activation of FER-mediated ROS generation in SI stigmas, ultimately resulting in UI pollen rejection (Figure 3c) [20]

However, it is still unclear if these mechanisms of pollen recognition by the stigma enhance the establishment of this interspecific/intergeneric barrier, as well as the degree of retention of these mechanisms in the plant species belonging to different families, including Solanaceae, actually exists (Figure 4c). Moreover, Lan et al. [122] showed that the pollen of distantly related species could only partially penetrate into the stigmata with a defective lock or after the pRALF26 treatment, suggesting the presence of some additional mechanisms. The secondary barrier for penetration can also serve as a rejection of Brassicaceae interspecific PTs. In total, seven leucine-rich repeats of Arabidopsis malectin receptor kinases (LRR-MAL RKs) have been found, in particular, RKF1, RKFL1-3, LysM RLK1-INTERACTING KINASE1, REMORIN-INTERACTING RECEPTOR1, and NEMATODE-INDUCED LRR-RLK2, which establish a prezygotic IRB for the Capsella rubella PTs during the penetration. As is reported, Arabidopsis PTs normally grew into the style; however, C. rubella PTs’ growth was hampered. This result demonstrates that the removal of the stigma barrier in the plants in which this barrier is the key one can lead to fertilization and development of hybrid embryos, thereby opening the way to successful interspecific/intergeneric hybridization. Summing up, the pRALF26 synthetic peptide successfully triggered a broader outcrossing and, thus, repeated a typical effect of pollen guidance. In general, these results clarify that the FER-CVY1/ANJ/HERK1-RALF complex, together with LRX proteins, represents the major molecular components of the pollen guidance in Brassicaceae [122] (Figure 3).

It should be noted that, according to the latest data, the RALF–FER mechanism is universal, fundamental, and tissue-specific in plant growth and response to abiotic stresses. For example, FER is involved in the regulation of auxin and abscisic acid responses, modulates growth and stress responses, controls cell wall integrity and ROS release in various tissues in response to RALF. It is also noted that the RALF–FER complex interferes with jasmonic acid signaling. Further study of the RALF–FER complex may shed light on hormonal regulation in IRB [122,123].

The stigma is the main site for the pollen interaction that specifies the interspecific/intergeneric barrier; the disruption of this stigma barrier is the crucial step in plant breeding and agriculture for achieving successful interspecific/intergeneric hybridization. In particular, the effect of PT guidance was discovered and accepted, but with limited success. However, this method also finds application in the current breeding of some plant species, for example, cactus [125]. Somoza et al. [126] finally discovered the mechanism underlying a more general interspecific/intergeneric reproductive barrier, shedding light on the PT guidance effect. The fact that the treatment of stigmata with paracrine pRALFs is able to repeat the effect of PT guidance will undoubtedly promote further application and improvement of this technology. It can also be extended to other plant species, enhancing interspecific/intergeneric crosses. Of interest is that the discovered fact that the PTs of distantly related species can only partially penetrate the stigmata with defective lock (as is observable in mutants fer, cvy1, and herk1, as well as ralf quad and lrx345) or after a pRALF26 treatment implies that additional components and/or mechanisms can also enhance the stigma reproductive barrier [38,127].

As for the monocots, the same function was ascribed to another small secreted protein, EA1 [128]. In both cases, PT attractants prevalently attract the PTs of the same species [61,128,129]. Each of these observations reflects the evolutionary pressure on this system so as to favor the crossing partners of the same species rather than interspecific hybrids.

Recent studies of the effect of nitric oxide (NO) on the compatible pollination in Arabidopsis have shown functional feedback between NO and ROS mediated by PCP-B and FER during this pollination. As was observed, the NO level on the stigma rapidly increased after the pollination, followed by its plummet to the initial level concurrently with pollen hydration. It is the presence of PCP-B and FER that influenced the increase in NO [130]. As a result, FER can serve as a target for NO nitrosation; in this process, the interaction of nitrosated FER with the downstream ROP2 is impaired, thereby interfering with ROS generation. However, the signaling mechanisms following these events on a cellular level remain vague [125].

5.3. PG Germination and PT Growth in IRBs

Interesting data on the PT germination in IRBs were obtained by a research team when studying the plants of the genus Quercus, the beech family [4]. In an intraspecific hybridization of Q. variabilis, PGs commence germination 6 h after pollination, and PTs enter the stigma in 24 h, versus the interspecific hybridization with Q. mongolica, when PGs germinate after 15 h. Intermittence is a sign of PT growth in Fagaceae as well, that is, their PTs can halt their growth at a number of cessation sites, including the style, upper part of the ovary, chalaza, and micropyle, which enhances the delay in fertilization [131]. Moreover, this pattern emerged to be common for the Quercus: PTs must be stopped for a long time interval at the interface between the stigma and style (12–13 months for Q. acutissima), and the style base is an effective filter preventing the growth of most PTs [132,133]. The anatomy suggests that the cessation sites in the pistil, namely at the style base and chalaza, are surrounded by vascular bundles [134,135]; presumably, the selection of PT cessation sites is associated with nutritional demands. Of interest is that Q. acutissima microsections demonstrate distinct histological differences between the upper wall of the ovary locule, a PT growth cessation site [135].

Similar studies have been performed with other objects. In particular, the PT growth in petunia pistils in a compatible conspecific pollination comprises two steps: a relatively slow growth during the first 8 h after pollination, followed by a rapid growth phase coinciding with the penetration into the style. The P. integrifolia PTs in the P. axillaris pistil grow more slowly, and the transition to fast growth is absent in this pollination type. A slow growth means that the flower senescence takes place earlier than the heterospecific PTs can reach the ovary. Consequently, the unsuccessful pollination in this cross (P. axillaris × P. integrifolia, with the former as a female plant) is regarded as a case of interspecific incompatibility [73,136].

In addition, the interaction between pollen and pistil is controlled by a complex molecular system that depends on the stigma structure and size, pollen hydration, pollen cover, specific proteins, and protein–protein interactions [137]. As is shown, these mechanisms are involved in the interspecific isolation by interfering with the PT guidance or targeting to the ovules [130]. However, many blind zones remain regarding the molecular components of these barriers, requiring further studies. It is known that the PTs of dicots respond to synergid control signals, in particular, LURE proteins, which guide the elongating PT tips to female gametes [138].

The disturbances in PT growth regulation and their targeting can appear in the absence of PT germination [139], PT lateral growth or deformation [140], PT burst [141], their growth slowdown and prevention of their entering the ovule [142], and the absence of fertilization even in the case when PTs reach the embryo sac. Even if PTs enter the ovary and reach the embryo sac, it can still remain unfertilized, or only the egg cell nucleus or polar nucleus performs a single fertilization [143].

Some evidence suggests that the hormonal signals and the corresponding receptors control the intercellular connection, playing an important role in the regulation of the progamic fertilization phase. During fertilization, the interaction between the female and male gametophytes, including the pollen–pistil and style–ovule recognition, to a considerable degree depends on the signaling pathways mediated by phytohormones. It is shown that the first hormonal signaling that regulates the pollen recognition by the stigma is that by ethylene [144] (Figure 3 and Figure 4). Other phytohormones are also involved in the function and regulation of IRBs. For example, an intergeneric hybridization of the wheat (Triticum aestivum L.) and maize (Zea mays L.) allows double haploids of wheat to be produced with a high efficiency in the case when auxin is applied to each individual wheat inflorescence 1 day after pollination. Approximately 2 weeks after crossing, in vitro embryo culture makes it possible to regenerate haploid wheat seedlings after eliminating the maize chromosomes [145]. The hormonal aspect of IRB regulation is an almost unstudied but most promising area for further research.

In our studies on the role of phytohormones in the gametophytic SI mechanism in P. hybrida, we showed that the growth arrest of self-incompatible pollen tubes occurs at elevated levels of ethylene and ABA in the stigma and cytokinins in the style. Moreover, we managed to overcome self-incompatibility by inhibiting ethylene—the pollen tubes grew to the ovary and formed seeds [144]. Possibly, treatments of the stigma with the ethylene inhibitor AOA may also affect the relief of IRB.

It is known that self-incompatibility triggers a signaling network involving rapid Ca²⁺ influx and increased cytosolic free calcium ([Ca²⁺]cyt). The increase in reactive oxygen species (ROS), sharp ATP depletion, and acidification of cytosolic pH ([pH]cyt) occur within 10 min after SI induction in Papaver. ATP depletion likely causes inactivation of the H⁺-ATPase pump. The activity of soluble inorganic pyrophosphatase (sPPase) is suppressed, leading to inhibition of cellular biosynthesis. Increased [Ca²⁺]cyt and ROS and decreased [pH]cyt also cause changes in the actin cytoskeleton, which rapidly undergoes fragmentation and depolymerization. All these very early events contribute to rapid inhibition of pollen tube tip growth within 1–2 min after SI initiation. Increased [Ca²⁺]cyt and ROS and decreased [pH]cyt signal several downstream targets to induce PCD. Cytosolic acidification is crucial for initiating PCD, since the caspase-3-like DEVDase enzyme is inactive at normal [pH]cyt. Activation of this enzyme several hours after signaling network initiation determines the fate of incompatible pollen, with commitment to cell suicide ensuring that SI-inhibited pollen tubes do not reach fertilization [146]. Similar processes are observed in the functioning of the SI mechanism in petunia (Figure 4b), but we found no data on the involvement of all these pollen tube growth arrest chain components in the functioning of IRB mechanisms in plants with wet stigmas.

Pease et al. [147] used transcriptome analysis to identify the candidate genes associated with the PT growth arrest. Five of the 20 genes best expressed in the pistil were associated with cell wall modification. One of these genes, found in S. pennellii, coding for pectin methylesterase inhibitor (PMEI), is absent in the genome of S. lycopersicum. It is known that the level of pectin methylation plays a key role in the normal PT growth. Demethylated pectin forms a solid gel in PT, providing the advance through the pistil transmitting tissue, while PMEI is mainly localized to the flexible and expanding PT part, where it regulates its growth direction [148]. A disturbed pollen formation and unstable PT growth were observed in the Arabidopsis carrying mutations in the PMEI and VGD1 genes [149]; and the addition of exogenous ZmPMEI1 from maize (Z. mays) causes their rupture during growth in vitro. Consequently, this suggests an important role of the S. pennellii LA0716 PMEI gene in the regulation of PT growth.

In addition to pectin methylesterases and their inhibitors, the transmitting tissues carry other cell matrix components, such as arabinogalactan proteins, enhancing the PT nutrition and growth in the pistil [150]. However, bioinformatics analysis suggests that these proteins can also be involved in the arrest of PT growth [16], although any direct evidence is still absent. Presumably, these proteins of Q. mongolica contribute to PT growth slowdown, which is of great interest for further studies.

5.4. PT Ingrowth into the Embryo Sac in IRBs

Synergid cells (Figure 2b,f) act as the final “doorkeepers” between the male and female gametes. Once the interaction between a PT and a receptive synergid cell is successful, both cells burst to release two sperm cells to the fertilization zone [151]. However, this burst does not frequently take place in interspecific crosses, suggesting that this stage serves as a prezygotic barrier preventing interspecific fertilization [152].

Genetic studies [153] detected the signaling pathway necessary to burst the PT and synergid cells in Arabidopsis. An impairment of the function of receptor kinase FERONIA (FER), expressed in synergid cells [151], or its coreceptor glycosylphosphatidylinositol (GPI) anchor protein LORELEI (LRE) [154] decreases the efficiency of PT and synergid cell burst, which is accompanied by a decrease in fertility. An analogous effect is observed with the loss of the function of transmembrane protein NORTIA (NTA), which acts as a calcium channel and migrates to the interface of PT and synergid under the control of FERONIA [155].

In addition, the HERCULES (HERK) and ANJEA (ANJ) double mutants belonging to the same family of C. roseus RLK-1–like kinase (CrRLK1L) as FERONIA, display similar impairments in sperm cell release and PT twisting as the feronia mutants but have no effect on the FER-dependent ROS generation at the PT–synergid interface [156]. These results confirm that FERONIA performs several functions at different stages in the interaction of PT and the synergid cell.

The genes expressed in PT are also important for its burst. A loss in the function of MYB transcription factors (myb97, myb101, and myb120) expressed in PT decreases the expression of their target genes and the efficiency of PT and synergid burst, thereby decreasing fertility [157].

The reciprocal crosses Abelmoschus manihot var. tetraphyllus × A. esculentus demonstrated a considerable decrease in the PT penetration into the ovule and pronounced PT deformations as compared with the direct cross (A. esculentus × A. manihot var. tetraphyllus), suggesting a strong rejection of A. esculentus pollen by the A. manihot var. tetraphyllus pistil. These results agree with the data on the pollen–pistil interaction in interspecific crosses [158]. Similar phenomena were observed in cotton and sesame [159]. These differences in PT development suggest the presence of prezygotic barriers acting at the level of the pistil. However, PTs in general developed normally in the selfed F1 and C1 pistils as well as in the reciprocal crossing direction (A. esculentus × F1/C1), but the in vivo pollen germination considerably decreased. Most likely, this decrease is associated with hybrid sterility rather than with prezygotic barriers preventing pollen germination or its growth in the stigma and style. The pollen–pistil interaction was normal in both direct and reciprocal crosses (F1/C1 × A. esculentus) [160].

Elegant work was carried out by Zenkteler and Relska-Roszak [161] on representatives of gymnosperms. In different gymnosperms, ovules receive pollen at different developmental stages, while the existing incompatibility mechanism acts later and occurs inside the ovule. Barriers to foreign pollen may arise during pollen germination, pollen penetration into the nucellus, and pollen penetration into the megagametophyte. In work concerning in vitro crosses among conifers, it was shown that megagametophyte cells produce secretions attractive for pollen development and growth into megagametophytes belonging to different genera. We managed to find only a few articles [8,161] regarding pollen germination of certain gymnosperm species on angiosperm placentas. These studies showed that gymnosperm pollen grains are capable of germinating shortly after placement on angiosperm ovules, and that pollen tubes sometimes entered the micropyle. Thus, barriers to angiosperm pollen may be absent on gymnosperm ovules/nucelli. However, discrimination of angiosperm pollen may occur in the pollination drop of the micropyle. As mentioned by Owens et al. [162], the pollination drop, rich in sugars, amino acids, peptides, and organic acids, inhibits germination of foreign pollen, thereby acting as one of the mechanisms of prezygotic incompatibility.

6. Overcoming IRBs

Interspecific gene introgression in nature is one of the driving forces in plant evolution. In addition, interspecific and intergeneric hybridization is regarded as the most important tool for increasing genetic variation and has been used for the breeding of many ornamental species. Correspondingly, the attempts to cross different cultivated species with one another or their wild relatives are widely used to obtain cultivars with new desirable traits; in this process, breeders have to overcome various IRBs [163]. That is why distant hybridization with its incompatibility, presenting a serious problem for researchers, can be overcome using several approaches, including pollination before flowering [164], pollination with a pollen mixture [165], repeated pollination [166], use of appropriate parents [167], and interspecific hybridization.

Currently, the researchers challenging to overcome IRBs follow several directions: (1) study of the effect of pollen guidance [125] in combination with γ-irradiation, X-rays, or chemical treatment for deactivating the mentor pollen before pollination [168,169]; (2) the search for the proteins and receptors in the stigma able to recognize foreign pollen [122]; (3) study of the hormonal balance and treatments with growth regulators in distant hybridization [170]; (4) duplication of chromosomes [171]; (5) attempts to link the SI phenomenon with reproductive barriers in distant hybridization [62,86]; (6) search for the candidate genes to describe reproductive barrier [155]; (7) study of hybrid necrosis [14]; (8) embryo rescue [171]; (9) in vitro pollination [172], Zenkteler [8] conducted important experiments showing successful germination of gymnosperm pollen (Ephedra distachya and Pinus wallichiana) on cultured angiosperm placentas (Nicotiana tabacum, Melandrium album, and Allium moly). In some crosses, they even observed gynogenetic haploid proembryos; (10) induction of unreduced gametes [173]; (11) protoplast fusion [174]; and some others. All these approaches are, to a certain degree, able to bypass prezygotic or postzygotic IRBs.

A combination of cytological knowledge (number of chromosomes, ploidy levels, and genome size) and phylogenetic data can predict the crossing efficiency or assist in making a careful decision on the combination of crosses. Genome editing systems can be helpful in the future in overcoming the existing crossing barriers and accelerating breeding strategies [175]. The results show that although distant hybridization is generally less efficient, hybridization success can be improved by using these advanced techniques [176].

7. Conclusions

Insight into this issue provides a clearer understanding of the underlying mechanisms and assists in designing the techniques that allow the problems associated with interspecific/intergeneric hybridization in biology and plant breeding to be resolved. The current understanding of how the interaction between PGs, PTs, and pistil tissues acts as IRBs is very limited. The recognition and key molecular mechanisms are still vague. The study of reproductive barriers is an important challenge and a most promising area for further research. The discovery of incompatibility mechanisms in the pollen–pistil system is a valuable tool for future breeding of agricultural crops.

Currently, a considerable advance in the analysis of molecular and some physiological aspects of the SI has not yet sufficiently clarified the function of signaling systems in IRBs. There are no data on the roles of phytohormones, ROS, and PCD in this process (Figure 3c and Figure 4c). What is necessary to overcome IRBs? Only a few methods allow the barriers to be surmounted, but the attempts to breed new highly productive and stable hybrids, in some cases, with improved ornamental characteristics, fail.

Note in conclusion that wide hybridization is promising for crop improvement. This approach, including the crossing of distantly related species, is able to reveal valuable genetic diversity and introduce new characters able to improve crop resistance, yield, nutritional value, and ornamental quality. The possibility to utilize the genetic potential of wide hybridization becomes ever more essential with increasing global challenges, such as climate change and population growth. Despite the existing obstacles and difficulties associated with this technique, the current studies and technological advances pave the way to its successful application.