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In Solanaceae, the self-incompatibility S-RNase and S-locus F-box interactions define self-pollen recognition and rejection in an S-specific manner. This interaction triggers a cascade of events involving other gene products unlinked to the S-locus that are crucial to the self-incompatibility response. To date, two essential pistil-modifier genes, 120K and High Top-Band (HT-B), have been identified in Nicotiana species. However, biochemistry and genetics indicate that additional modifier genes are required. We recently reported a Kunitz-type proteinase inhibitor, named NaStEP (for Nicotiana alata Stigma-Expressed Protein), that is highly expressed in the stigmas of self-incompatible Nicotiana species. Here, we report the proteinase inhibitor activity of NaStEP. NaStEP is taken up by both compatible and incompatible pollen tubes, but its suppression in Nicotiana spp. transgenic plants disrupts S-specific pollen rejection; therefore, NaStEP is a novel pistil-modifier gene. Furthermore, HT-B levels within the pollen tubes are reduced when NaStEP-suppressed pistils are pollinated with either compatible or incompatible pollen. In wild-type self-incompatible N. alata, in contrast, HT-B degradation occurs preferentially in compatible pollinations. Taken together, these data show that the presence of NaStEP is required for the stability of HT-B inside pollen tubes during the rejection response, but the underlying mechanism is currently unknown. [ABSTRACT FROM AUTHOR]

Background S-RNase-based self-incompatibility (SI) occurs in the Solanaceae, Rosaceae and Plantaginaceae. In all three families, compatibility is controlled by a polymorphic S-locus encoding at least two genes. S-RNases determine the specificity of pollen rejection in the pistil, and S-locus F-box proteins fulfill this function in pollen. S-RNases are thought to function as S-specific cytotoxins as well as recognition proteins. Thus, incompatibility results from the cytotoxic activity of S-RNase, while compatible pollen tubes evade S-RNase cytotoxicity. Scope The S-specificity determinants are known, but many questions remain. In this review, the genetics of SI are introduced and the characteristics of S-RNases and pollen F-box proteins are briefly described. A variety of modifier genes also required for SI are also reviewed. Mutations affecting compatibility in pollen are especially important for defining models of compatibility and incompatibility. In Solanaceae, pollen-side mutations causing breakdown in SI have been attributed to the heteroallelic pollen effect, but a mutation in Solanum chacoense may be an exception. This has been interpreted to mean that pollen incompatibility is the default condition unless the S-locus F-box protein confers resistance to S-RNase. In Prunus, however, S-locus F-box protein gene mutations clearly cause compatibility. Conclusions Two alternative mechanisms have been proposed to explain compatibility and incompatibility: compatibility is explained either as a result of either degradation of non-self S-RNase or by its compartmentalization so that it does not have access to the pollen tube cytoplasm. These models are not necessarily mutually exclusive, but each makes different predictions about whether pollen compatibility or incompatibility is the default. As more factors required for SI are identified and characterized, it will be possible to determine the role each process plays in S-RNase-based SI. [ABSTRACT FROM PUBLISHER]

The endomembrane system offers many potential points where plant mating can be effectively controlled. This results from two basic features of angiosperm reproduction: the requirement for pollen tubes to pass through sporophytic tissues to gain access to ovules and the physiology of pollen tube growth that provides it with the capacity to do so. Rapid pollen tube growth requires extravagant exocytosis and endocytosis activity as cell wall material is deposited and membrane is recovered from the actively growing tip. Moreover, recent results show that pollen tubes take up a great deal of material from the pistil extracellular matrix. Regarding the stigma and style as organs specialized for mate selection focuses attention on their complementary roles in secreting material to support the growth of compatible pollen tubes and discourage the growth of undesirable pollen. Since these processes also involve regulated activities of the endomembrane system, the potential for regulating mating by controlling endomembrane events exists in both pollen and pistil. [ABSTRACT FROM PUBLISHER]

Charles Darwin made extensive observations of the pollination biology of a wide variety of plants. He carefully documented the consequences of self-pollination and described species that were self-sterile but that could easily be crossed with other plants of the same species. He believed that compatibility was controlled by the ‘mutual action’ of pollen and pistil contents. A genetic model for self-sterility was developed in the early 1900s based on studies of the compatibility relationships among, what are now referred to as, self-incompatible (SI) Nicotiana species. Today, it is believed that SI in these species is controlled by an interaction between S-RNases produced in the pistil and F-box proteins expressed in pollen and, moreover, that this S-RNase-based SI system is shared by a great diversity of other plant species. Current research is aimed at understanding how the mutual actions of these S-gene products function in the physiological context of pollen tube growth. [ABSTRACT FROM PUBLISHER]

After landing on a wet stigma, pollen grains hydrate and germination generally occurs. However, there is no certainty of the pollen tube growth through the style to reach the ovary. The pistil is a gatekeeper that evolved in many species to recognize and reject the self-pollen, avoiding endogamy and encouraging cross-pollination. However, recognition is a complex process, and specific factors are needed. Here the isolation and characterization of a stigma-specific protein from N. alata, NaStEP (N. alata Stigma Expressed Protein), that is homologous to Kunitz-type proteinase inhibitors, are reported. Activity gel assays showed that NaStEP is not a functional serine proteinase inhibitor. Immunohistochemical and protein blot analyses revealed that NaStEP is detectable in stigmas of self-incompatible (SI) species N. alata, N. forgetiana, and N. bonariensis, but not in self-compatible (SC) species N. tabacum, N. plumbaginifolia, N. benthamiana, N. longiflora, and N. glauca. NaStEP contains the vacuolar targeting sequence NPIVL, and immunocytochemistry experiments showed vacuolar localization in unpollinated stigmas. After self-pollination or pollination with pollen from the SC species N. tabacum or N. plumbaginifolia, NaStEP was also found in the stigmatic exudate. The synthesis and presence in the stigmatic exudate of this protein was strongly induced in N. alata following incompatible pollination with N. tabacum pollen. The transfer of NaStEP to the stigmatic exudate was accompanied by perforation of the stigmatic cell wall, which appeared to release the vacuolar contents to the apoplastic space. The increase in NaStEP synthesis after pollination and its presence in the stigmatic exudates suggest that this protein may play a role in the early pollen–stigma interactions that regulate pollen tube growth in Nicotiana. [ABSTRACT FROM PUBLISHER]

HT-family proteins have been identified in Nicotiana, Solanum, and Petunia. HT-B-type proteins are implicated in S-RNase-based self-incompatibility, but the functions of other family members are unknown. Screening for cDNA sequences with an expression pattern similar to HT-B in Nicotiana alata revealed a new group of small HT-family proteins, designated HT-M. HT-M proteins resemble HT-B in several respects: their pistil-specific expression pattern is indistinguishable from HT-B, they pellet with a microsome fraction, and their abundance decreases after pollination. Unlike HT-B, there is no S-specificity to this response, and RNAi experiments show that HT-M proteins are not necessary for self-incompatibility. Identification of a third group of pistil-specific HT-family proteins helps better define the characteristics of the family and allowed identification of putative new family members. By searching the databases with only the most conserved HT-family sequence elements, the signal sequence and cysteine motifs, we identified nodulin-24-like proteins and several small glycine-rich proteins as putative HT-family members. Like HT-M and HT-B, nodulin-24 is membrane associated. We propose that the conserved features in HT-family proteins are important for targeting or modification and refer to the broader family that includes both HT- and nodulin-24-like proteins as the HT/NOD-24-family. [ABSTRACT FROM PUBLISHER]

S-RNase-based self-incompatibility (SI) is the most widespread form of genetically controlled mate selection in plants. S-RNase controls pollination specificity in the pistil, while the newly discovered SLF/SFB controls pollination specificity in the pollen. A widely discussed model suggests that compatibility is explained by ubiquitylation and degradation of nonself-S-RNase and that, conversely, incompatibility is caused by failure to degrade self-S-RNase. This model is consistent with the long-standing view that S-RNase inhibition is central to SI. Recent results show, however, that S-RNase is compartmentalized in pollen tubes and, significantly, that compatibility might not require SLF/SFB. S-RNase compartmentalization and dislocation into the pollen tube cytoplasm might be similar to the trafficking of other cytotoxins such as ricin. [Copyright &y& Elsevier]

Thioredoxins type h are classified into three subgroups. The sub-group II includes thioredoxins containing an N-terminal extension, the role of which is still unclear. Although thioredoxin secretion has been observed in animal cells, there is no evidence suggesting that any thioredoxin h is secreted in plants. In this study, we report that a thioredoxin h, subgroup II, from Nicotiana alata (NaTrxh) is secreted into the extracellular matrix of the stylar transmitting tract tissue. Fractionation studies showed that NaTrxh is extracted along with well characterized secretion proteins such as S-RNases and NaTTS (N. alata transmitting tissue-specific protein). Moreover, an NaTrxh-green fluorescent fusion protein transiently expressed in Nicotiana benthamiana and Arabidopsis thaliana leaves was also secreted, showing that NaTrxh has the required information for its secretion. We performed reduction assays in vitro to identify potential extracellular targets of NaTrxh. We found that S-RNase is one of the several potential substrates of the NaTrxh in the extracellular matrix. In addition, we proved by affinity chromatography that NaTrxh specifically interacts with S-RNase. Our findings showed that NaTrxh is a new thioredoxin h in Nicotiana that is secreted as well as in animal systems. Because NaTrxh is localized in the extracellular matrix of the stylar transmitting tract and its specific interaction with S-RNase to reduce it in vitro, we suggest that this thioredoxin h may be involved either in general pollen-pistil interaction processes or particularly in S-RNase-based self-incompatibility. [ABSTRACT FROM AUTHOR]

Determines the identification of pollen side (pollen S) in several plants. Genetics of self-compatibility (SI); Mechanism of S-RNase-based Si; Role of f-box proteins in determining S-specificity in pollen; S-haplotype interactions in pollen.

Focuses on a study aimed to identify non-S-RNase sequences that are required for pollen rejection. Materials & methods; Results.

Studies the ribosomal RNA (rRNA) gene structure of large red algal order Gigartinales in Southern Australia. Amplification of the rRNA; Site of rRNA gene; Size of the ribosomal DNA and its flanking internal transcribed spacers within and between genera.

Summary In a new study, the Papaver rhoeas (poppy family) self-incompatibility system has been transferred into Arabidopsis thaliana , a distantly related plant with a very different floral structure. The simple poppy self-incompatibility system may finally make it possible to introduce this potentially valuable trait into any plant. [ABSTRACT FROM AUTHOR]

Focuses on the identification of mating systems in flowering plants that determine whether a pollen suitor is accepted or rejected. Effect of mate selection on a species; Evolution of sophisticated mating control systems that require communication between pollen; Production of pollen tube to carry the haploid sperm cells.

Plants have mechanisms to recognize and reject pollen from other species. Although widespread, these mechanisms are less well understood than the self-incompatibility ( SI) mechanisms plants use to reject pollen from close relatives. Previous studies have shown that some interspecific reproductive barriers ( IRBs) are related to SI in the Solanaceae. For example, the pistil SI proteins S- RNase and HT protein function in a pistil-side IRB that causes rejection of pollen from self-compatible ( SC) red/orange-fruited species in the tomato clade. However, S- RNase-independent IRBs also clearly contribute to rejecting pollen from these species. We investigated S- RNase-independent rejection of Solanum lycopersicum pollen by SC Solanum pennellii LA0716, SC. Solanum habrochaites LA0407, and SC Solanum arcanum LA2157, which lack functional S- RNase expression. We found that all three accessions express HT proteins, which previously had been known to function only in conjunction with S- RNase, and then used RNAi to test whether they also function in S- RNase-independent pollen rejection. Suppressing HT expression in SC S. pennellii LA0716 allows S. lycopersicum pollen tubes to penetrate farther into the pistil in HT suppressed plants, but not to reach the ovary. In contrast, suppressing HT expression in SC. Solanum habrochaites LA0407 and in SC S. arcanum LA2157 allows S. lycopersicum pollen tubes to penetrate to the ovary and produce hybrids that, otherwise, would be difficult to obtain. Thus, HT proteins are implicated in both S- RNase-dependent and S- RNase-independent pollen rejection. The results support the view that overall compatibility results from multiple pollen-pistil interactions with additive effects. [ABSTRACT FROM AUTHOR]

Summary: Expressing a pollen self-incompatibility gene from Papaver rhoeas (poppy) in Arabidopsis thaliana renders the latter sensitive to an exquisitely precise induced cell death response. This simple system may have wide application in biotechnology and research. [ABSTRACT FROM AUTHOR]

A preface to the volume 61, number 7 issue of the "Journal of Experimental Botany" is presented.

The tomato clade within the genus Solanum has numerous advantages for mechanistic studies of reproductive isolation. Its thirteen closely related species, along with four closely allied Solanum species, provide a defined group with diverse mating systems that display complex interspecific reproductive barriers. Several kinds of pre- and postzygotic barriers have already been identified within this clade. Well-developed genetic maps, introgression lines, interspecific bridging lines, and the newly available draft genome sequence of the domesticated tomato ( Solanum lycopersicum) are valuable tools for the genetic analysis of interspecific reproductive barriers. The excellent chromosome morphology of these diploid species allows detailed cytological analysis of interspecific hybrids. Transgenic methodologies, well developed in the Solanaceae, allow the functional testing of candidate reproductive barrier genes as well as live imaging of pollen rejection events through the use of fluorescently tagged proteins. Proteomic and transcriptomics approaches are also providing new insights into the molecular nature of interspecific barriers. Recent progress toward understanding reproductive isolation mechanisms using these molecular and genetic tools is assessed in this review. [ABSTRACT FROM AUTHOR]

As pollen tubes grow toward the ovary, they are in constant contact with the pistil extraceilular matrix (ECM). ECM components are taken up during growth, and some pistil molecules exert their effect inside the pollen tube. For instance, the Nicotiana alata 120-kD glycoprotein (120K) is an abundant arabinogalactan protein that is taken up from the ECM; it has been detected in association with pollen tube vacuoles, but the transport pathway between these compartments is unknown. We recently identified a pollen C2 domain-containing protein (NaPCCP) that binds to the carboxyl-terminal domain of 120K. As C2 domain proteins mediate protein-lipid interactions) NaPCCP could function in intracellular transport of 120K in pollen tubes. Here, we describe binding studies showing that the NaPCCP C2 domain is functional and that binding is specific for phosphatidylinositol 3-phosphate. Subcellular fractionation, immunolocalization, arid live imaging results show that NaPCCP is associated with the plasma membrane and internal pollen tube vesicles. Colocalization between an NaPCCP::green fluorescent protein fusion and internalized FM4-64 suggest an association with the endosomal system. NaPCCIP localization is altered in pollen tubes rejected by the self-incompatibility mechanism, but our hypothesis is that it has a general function in the transport of endocytic cargo rather than a specific function in self-incompatibility. NaPCCP represents a bifunctional protein with both phosphatidylinositol 3-phosphate- and arabirnogalactan protein-binding domains. Therefore, it could function in the transport of pistil ECM proteins in the pollen tube endomembrane system. [ABSTRACT FROM AUTHOR]

Pollen tube growth is influenced by interaction between pollen proteins and the pistil extracellular matrix. The transmitting tract-specific glycoprotein (NaTTS) and 120-kDa glycoprotein (120K) are two pistil arabinogalactan proteins (AGPs) that share a conserved C-terminal domain (CTD) and directly influence pollen tubes in Nicotiana alata. 120K and other extracellular matrix proteins are taken up and transported to vacuoles of growing pollen tubes. We hypothesize that signaling and trafficking processes inside pollen tubes are important for controlling pollen tube growth. We performed a yeast two-hybrid screen of pollen cDNAs using sequences from 120K and NaTTS as baits. We found that an S-RNase-binding protein (SBP1), a C2 domain-containing protein (NaPCCP), and a putative cysteine protease bound to the AGP baits. SBP1 from Petunia hybrida and Solanum chacoense is a putative E3 ubiquitin ligase that binds to S-RNase and other proteins. C2 domain-containing proteins bind lipids and can regulate myriad cellular processes. Cysteine proteases are often associated with the degradation of vacuolar proteins. Expression analysis revealed that transcripts for these proteins are expressed in mature pollen. NaPCCP and NaSBP1 were characterized further because of their potential roles in signaling and trafficking. In vitro pull-down assays verified binding between maltose-binding protein (MBP) fusions, MBP::NaPCCP or MBP::NaSBP1 and glutathione S-transferase (GST), GST::AGPCTD fusions. NaSBP1 binds to the AGP CTDs through its helical and RING domains. NaPCCP binds through its C-terminal region. Binding between NaPCCP and NaSBP1 and the pistil AGPs may contribute to signaling and trafficking inside pollen tubes growing in planta. [ABSTRACT FROM AUTHOR]

Nicotiana section Alatae contains eight species with variable flower sizes and morphologies. Section members readily hybridize in the glasshouse, but no hybrids have been observed in natural sympatric and parapatric populations. To investigate interspecific crossing relationships with respect to mechanisms preventing hybridization, all members of section Alatae were intercrossed in a complete diallel. We found positive correlation between the pistil length of the pollen donor and interspecific seed set relative to the conspecific cross. Pollen tube growth rate and pollen donor pistil length were positively correlated as well. Furthermore, pollen from short-pistil members of section Alatae could only grow a maximum distance proportional to, but greater than, their own pistil lengths. Our results show that pollen tube growth capacity (i.e., rate and distance), provides a hybridization barrier in long-pistil species × short-pistil species crosses. We also found another hybridization barrier not specifically related to pollen tube growth capacity in short-pistil species × long-pistil species. Taken together, these barriers can generally be described by a ‘pistil-length mismatch’ rule; in section Alatae, pollen has the most success fertilizing ovules from species with pistil lengths similar to their own. This rule could contribute to hybridization barriers in Section Alatae because the species display dramatically different pistil lengths. [ABSTRACT FROM AUTHOR]

S-RNase participates in at least three mechanisms of pollen rejection. It functions in S-specific pollen rejection (self-incompatibility) and in at least two distinct interspecific mechanisms of pollen rejection in Nicotiana. S-specific pollen rejection and rejection of pollen from Nicotiana plumbaginifolia also require additional stylar proteins. Transmitting-tract-specific (TTS) protein, 120 kDa glycoprotein (120K) and pistil extensin-like protein III (PELP III) are stylar glycoproteins that bind S-RNase in vitro and are also known to interact with pollen. Here we tested whether these glycoproteins have a direct role in pollen rejection. 120K shows the most polymorphism in size between Nicotiana species. Larger 120K-like proteins are often correlated with S-specific pollen rejection. Sequencing results suggest that the polymorphism primarily reflects differences in glycosylation, although indels also occur in the predicted polypeptides. Using RNA interference (RNAi), we suppressed expression of 120K to determine if it is required for S-specific pollen rejection. Transgenic SC N. plumbaginifolia × SI Nicotiana alata ( S105 S105 or SC10 SC10) hybrids with no detectable 120K were unable to perform S-specific pollen rejection. Thus, 120K has a direct role in S-specific pollen rejection. However, suppression of 120K had no effect on rejection of N. plumbaginifolia pollen. In contrast, suppression of HT-B, a factor previously implicated in S-specific pollen rejection, disrupts rejection of N. plumbaginifolia pollen. Thus, S-specific pollen rejection and rejection of N. plumbaginifolia pollen are mechanistically distinct, because they require different non-S-RNase factors. [ABSTRACT FROM AUTHOR]

Biochemical interactions between the pollen and the pistil allow plants fine control over fertilization. S‐RNase‐based pollen rejection is among the most widespread and best understood of these interactions. At least three plant families have S‐RNase‐based self‐incompatibility (SI) systems, and S‐RNases have also been implicated in interspecific pollen rejection. Although S‐RNases determine the specificity of SI, other genes are required for the pollen rejection system to function. Progress is being made toward identifying these non‐S‐RNase factors. HT‐protein, first identified as a non‐S‐RNase factor that was required for SI in Nicotiana alata, has now been implicated in other species as well. In addition, several pistil proteins bind to S‐RNase in vitro. One hypothesis is that S‐RNase forms a complex with these proteins in vivo that is the active form of S‐RNase in pollen rejection. [ABSTRACT FROM PUBLISHER]

Summary: Mating system transitions from self‐incompatibility (SI) to self‐compatibility (SC) are common in plants. In the absence of high levels of inbreeding depression, SC alleles are predicted to spread due to transmission advantage and reproductive assurance.We characterized mating system and pistil‐expressed SI factors in 20 populations of the wild tomato species Solanum habrochaites from the southern half of the species range.We found that a single SI to SC transition is fixed in populations south of the Rio Chillon valley in central Peru. In these populations, SC correlated with the presence of the hab‐6 S‐haplotype that encodes a low activity S‐RNase protein. We identified a single population segregating for SI/SC and hab‐6. Intrapopulation crosses showed that hab‐6 typically acts in the expected codominant fashion to confer SC. However, we found one specific S‐haplotype (hab‐10) that consistently rejects pollen of the hab‐6 haplotype, and results in SI hab‐6/hab‐10 heterozygotes.We suggest that the hab‐10 haplotype could act as a genetic mechanism to stabilize mixed mating in this population by presenting a disadvantage for the hab‐6 haplotype. This barrier may represent a mechanism allowing for the persistence of SI when an SC haplotype appears in or invades a population. [ABSTRACT FROM AUTHOR]

Summary: In self‐incompatible Solanaceae, the pistil protein S‐RNase contributes to S‐specific pollen rejection in conspecific crosses, as well as to rejecting pollen from foreign species or whole clades. However, S‐RNase alone is not sufficient for either type of pollen rejection. We describe a thioredoxin (Trx) type h from Nicotiana alata, NaTrxh, which interacts with and reduces S‐RNase in vitro. Here, we show that expressing a redox‐inactive mutant, NaTrxhSS, suppresses both S‐specific pollen rejection and rejection of pollen from Nicotiana plumbaginifolia. Biochemical experiments provide evidence that NaTrxh specifically reduces the Cys155‐Cys185 disulphide bond of SC10‐Rnase, resulting in a significant increase of its ribonuclease activity. This reduction and increase in S‐RNase activity by NaTrxh helps to explain why S‐RNase alone could be insufficient for pollen rejection. Significance Statement: S‐RNase is very well known for its role in S‐RNase‐based self‐incompatibility and in some types of interspecific pollen rejection. Through a dominant‐negative approach, we show that a thioredoxin (Trx) type h from Nicotiana alata, NaTrxh, is required for both types of pollen rejection. This is a significant advance, although what sets our new contribution apart is that we show that NaTrxh reduces an S‐RNase disulphide and increases its activity, providing a remarkable level of insight into the biochemical mechanism of S‐RNase‐dependent pollen rejection. [ABSTRACT FROM AUTHOR]

A goal of speciation genetics is to understand how the genetic components underlying interspecific reproductive barriers originate within species. Unilateral incompatibility (UI) is a postmating prezygotic barrier in which pollen rejection in the female reproductive tract (style) occurs in only one direction of an interspecific cross. Natural variation in the strength of UI has been observed among populations within species in the wild tomato clade. In some cases, molecular loci underlying self-incompatibility (SI) are associated with this variation in UI, but the mechanistic connection between these intra- and inter-specific pollen rejection behaviors is poorly understood in most instances. We generated an F2 population between SI and SC genotypes of a single species, Solanum pennellii , to examine the genetic basis of intraspecific variation in UI against other species, and to determine whether loci underlying SI are genetically associated with this variation. We found that F2 individuals vary in the rate at which UI rejection occurs. One large effect QTL detected for this trait co-localized with the SI-determining S -locus. Moreover, individuals that expressed S-RNase—the S- locus protein involved in SI pollen rejection—in their styles had much more rapid UI responses compared with those without S-RNase protein. Our analysis shows that intraspecific variation at mate choice loci—in this case at loci that prevent self-fertilization—can contribute to variation in the expression of interspecific isolation, including postmating prezygotic barriers. Understanding the nature of such intraspecific variation can provide insight into the accumulation of these barriers between diverging lineages. [ABSTRACT FROM AUTHOR]

In plants, transitions in mating system from outcrossing to self-fertilization are common; however, the impact of these transitions on interspecific and interpopulation reproductive barriers is not fully understood. We examined the consequences of mating system transition for reproductive barriers in 19 populations of the wild tomato species Solanum habrochaites., We identified S. habrochaites populations with self-incompatible (SI), self-compatible (SC) and mixed population (MP) mating systems, and characterized pollen-pistil interactions among S. habrochaites populations and between S. habrochaites and other tomato species. We examined the relationship between mating system, floral morphology, interspecific and interpopulation compatibility and pistil SI factors., We documented five distinct phenotypic groups by combining reproductive behavior with molecular data. Transitions from SI to MP were not associated with weakened interspecific reproductive barriers or loss of known pistil SI factors. However, transitions to SC at the northern range margin were accompanied by loss of S-RNase, smaller flowers, and weakened (or absent) interspecific pollen−pistil barriers. Finally, we identified a subset of SC populations that exhibited a partial interpopulation reproductive barrier with central SI populations., Our results support the hypothesis that shifts in mating system, followed by additional loss-of-function mutations, impact reproductive barriers within and between species. [ABSTRACT FROM AUTHOR]

PREMISE OF THE STUDY: Interspecific reproductive barriers (IRBs) often prevent hybridization between closely related species in sympatry. In the tomato clade ( Solanum section Lycopersicon ), interspecific interactions between natural sympatric populations have not been evaluated previously. In this study, we assessed IRBs between members of the tomato clade from nine sympatric sites in Peru. METHODS: Coflowering was assessed at sympatric sites in Peru. Using previously collected seeds from sympatric sites in Peru, we evaluated premating prezygotic (floral morphology), postmating prezygotic (pollen-tube growth), and postzygotic barriers (fruit and seed development) between sympatric species in common gardens. Pollen-tube growth and seed development were examined in reciprocal crosses between sympatric species. KEY RESULTS: We confirmed coflowering of sympatric species at five sites in Peru. We found three types of postmating prezygotic IRBs during pollen-pistil interactions: (1) unilateral pollen-tube rejection between pistils of self-incompatible species and pollen of self-compatible species; (2) potential conspecific pollen precedence in a cross between two self-incompatible species; and (3) failure of pollen tubes to target ovules. In addition, we found strong postzygotic IRBs that prevented normal seed development in 11 interspecific crosses, resulting in seed-like structures containing globular embryos and aborted endosperm and, in some cases, overgrown endothelium. Viable seed and F1 hybrid plants were recovered from three of 19 interspecific crosses. CONCLUSIONS: We have identified diverse prezygotic and postzygotic IRBs that would prevent hybridization between sympatric wild tomato species, but interspecific hybridization is possible in a few cases. [ABSTRACT FROM AUTHOR]

premise of the STUDY: Self-incompatibility (SI) is a mechanism that prevents inbreeding in many plant species. The mutational breakdown of SI occurs frequently, yet relatively little is known about the evolutionary steps involved in the progressive loss of pistil and pollen SI function. METHODS : In Solanaceae, SI is the S-RNase-based gametophytic type. We used SI and SC populations of the wild tomato species Solarium habrochaites to study natural variation for two pollen SI factors: a Cullinl (CUL1) protein and an S-locus F-box protein (SLF-23). Pollen compatibility was assessed on an allotriploid tester line encoding an S-RNase recognized by SLF-23. Both pollen factors are required for compatibility on this tester line. Complementation tests and gene sequencing were used to identify mutations in CUL1 or SLF-23. KEY RESULTS: We detected loss-of-function mutations in CUL1 and/or SLF-23 in SC populations collected near the northern and southern geographic margins of this taxon's natural range. Nonmarginal SC and all SI accessions expressed mostly functional alleles of these pollen factors. Comparison of the CUL 1 sequences identified several shared deletion mutations present in both northern and southern margin SC accessions.CONCLUSIONS: Loss-of-function mutations in CUL1 and SLF-23 likely became fixed relatively late during SI to SC transitions, after loss of pistil SI function. Mutations in CUL1 establish unilateral incompatibility with SI populations and strengthen reproductive isolation. Point mutations common to northern and southern SC biotypes likely derive from shared ancestral variants found in more central SI populations. [ABSTRACT FROM AUTHOR]

* Premise of the study: Interspecific reproductive barriers (IRBs) act to ensure species integrity by preventing hybridization. Previous studies on interspecific crosses in the tomato clade have focused on the success of fruit and seed set. The SI x SC rule (SI species x SC species crosses are incompatible, but the reciprocal crosses are compatible) often applies to interspecific crosses. Because SI systems in the Solanaceae affect pollen tube growth, we focused on this process in a comprehensive study of interspecific crosses in the tomato clade to test whether the SI x SC rule was always followed. * Methods: Pollen tube growth was assessed in reciprocal crosses between all 13 species of the tomato clade using fluorescence microscopy. * Key results: In crosses between SC and SI species, pollen tube growth follows the SI x SC rule: interspecific pollen tube rejection occurs when SI species are pollinated by SC species, but in the reciprocal crosses (SC x SI), pollen tubes reach ovaries. However, pollen tube rejection occurred in some crosses between pairs of SC species, demonstrating that a fully functional SI system is not necessary for pollen tube rejection in interspecific crosses. Further, gradations in the strength of both pistil and pollen IRBs were revealed in interspecific crosses using SC populations of generally SI species. * Conclusion: The SI x SC rule explains many of the compatibility relations in the tomato clade, but exceptions occur with more recently evolved SC species and accessions, revealing differences in strength of both pistil and pollen IRBs. [ABSTRACT FROM AUTHOR]

Double fertilization in flowering plants requires the delivery of two immotile sperm cells to the female gametes by a pollen tube, which perceives guidance cues, modifies its tip growth direction, and eventually enters the micropyle of the ovule. In spite of the recent progress, so far, little is known about the signaling events in pollen tubes in response to the guidance cues. Here, we show that MPK3 and MPK6, two Arabidopsis (Arabidopsis thaliana) mitogen-activated protein kinases, mediate the guidance response in pollen tubes. Genetic analysis revealed that mpk3 mpk6 double mutant pollen has reduced transmission. However, direct observation of mpk3-/- mpk6-/- mutant pollen phenotype was hampered by the embryo lethality of double homozygous mpk3 mpk6 plants. Utilizing a fluorescent reporter-tagged complementation method, we showed that the mpk3 mpk6 mutant pollen had normal pollen tube growth but impaired pollen tube guidance. In vivo pollination assays revealed that the mpk3 mpk6 mutant pollen tubes were defective in the funicular guidance phase. By contrast, semi-in vitro guidance assay showed that the micropylar guidance of the double mutant pollen tube was normal. Our results provide direct evidence to support that the funicular guidance phase of the pollen tube requires an in vivo signaling mechanism distinct from the micropyle guidance. Moreover, our finding opened up the possibility that the MPK3/MPK6 signaling pathway may link common signaling networks in plant stress response and pollen-pistil interaction. [ABSTRACT FROM AUTHOR]

Interspecific reproductive barriers are poorly understood, but are central to the biological species concept. The pre-zygotic barriers between red- and green-fruited species in the tomato clade of the genus Solanum provide a model to better understand these barriers in plants. Compatibility usually follows the SI x SC rule: pollen from self-compatible ( SC) red-fruited species is rejected on pistils of the predominantly self-incompatible ( SI) green-fruited species, but the reciprocal crosses are compatible. This suggests that the interspecific reproductive barrier may be linked to the intraspecific SI mechanism. However, pollen from the SC red-fruited species is also rejected by SC accessions of green-fruited species that lack S- RNase, a key protein expressed in pistils of SI Solanum species. Thus, multiple mechanisms may contribute to the barrier between red- and green-fruited species. We tested whether an S- RNase-dependent barrier is sufficient for rejection of pollen from red-fruited species by introducing functional S- RNase, HT- A and HT- B genes from SI species into Solanum lycopersicum (cultivated tomato). We found that expressing S- RNase in combination with either HT- A or HT- B in the pistil is sufficient to cause rejection of pollen from all four red-fruited species. Thus, redundant mechanisms must operate side by side to prevent crosses between red- and green-fruited species in the clade, underlining the complexity of interspecific pollination barriers. Our results also have implications for mating system transitions. We suggest that these transitions must occur in a specific sequence, and that the transition from SI to SC also affects interspecific compatibility. [ABSTRACT FROM AUTHOR]

Although self-incompatibility (SI) in plants has been studied extensively, far less is known about interspecific reproductive barriers. One interspecific barrier, known as unilateral incongruity or incompatibility (UI), occurs when species display unidirectional compatibility in interspecific crosses. In the wild tomato species Solanum pennellii, both SI and self-compatible (SC) populations express UI when crossed with domesticated tomato, offering a useful model system to dissect the molecular mechanisms involved in reproductive barriers. In this study, the timing of reproductive barrier establishment during pistil development was determined in SI and SC accessions of 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 prior to flower opening, but were established by 2 days before flower opening, with partial barriers detected during a transition period 3–4 days before flower opening. The developmental expression dynamics of known SI factors, S-RNases and HT proteins, was also examined. The accumulation of HT-A protein coincided temporally and spatially with UI barriers in developing pistils. Proteomic analysis of stigma/styles from key developmental stages showed a switch in protein profiles from cell-division-associated proteins in immature stigma/styles to a set of proteins in mature stigma/styles that included S-RNases, HT-A protein and proteins associated with cell-wall loosening and defense responses, which could be involved in pollen–pistil interactions. Other prominent proteins in mature stigma/styles were those involved in lipid metabolism, consistent with the accumulation of lipid-rich material during pistil maturation. [ABSTRACT FROM AUTHOR]

Wild tomato species in Solanum Section Lycopersicon often exhibit two types of reproductive barriers: self-incompatibility (SI) and unilateral incompatibility or incongruity (UI), wherein the success of an inter-specific cross depends on the direction of the cross. UI pollen rejection often follows the 'SI × SC' rule, i.e. pistils of SI species reject the pollen of SC (self-compatible) species but not vice versa, suggesting that the SI and UI pollen rejection mechanisms may overlap. In order to address this question, pollen tube growth was measured after inter-specific crosses using wild tomato species as the female parents and pollen from cultivated tomato ( Solanum lycopersicum). Two modes of UI pollen rejection, early and late, were observed, and both differed from SI pollen rejection. The structure and expression of known stylar SI genes were evaluated. We found that S-RNase expression is not required for either the early or late mode of UI pollen rejection. However, two HT family genes, HT-A and HT-B, map to a UI QTL. Surprisingly, we found that a gene previously implicated in SI, HT-B, is mutated in both SI and SC S. habrochaites accessions, and no HT-B protein could be detected. HT-A genes were detected and expressed in all species examined, and may therefore function in both SI and UI. We conclude that there are significant differences between SI and UI in the tomato clade, in that pollen tube growth differs between these two rejection systems, and some stylar SI factors, including S-RNase and HT-B, are not required for UI. [ABSTRACT FROM AUTHOR]

Tomato clade species (Solanum sect. Lycopersicon) display multiple interspecific reproductive barriers (IRBs). Some IRBs conform to the SI x SC rule, which describes unilateral incompatibility (UI) where pollen from SC species is rejected on SI species' pistils, but reciprocal pollinations are successful. However, SC x SC UI also exists, offering opportunities to identify factors that contribute to S-RNase-independent IRBs. For instance, SC Solanum pennellii LA0716 pistils only permit SC Solanum lycopersicum pollen tubes to penetrate to the top third of the pistil, while S. pennellii pollen penetrates to S. lycopersicum ovaries. We identified candidate S. pennellii LA0716 pistil barrier genes based on expression profiles and published results. CRISPR/Cas9 mutants were created in eight candidate genes, and mutants were assessed for changes in S. lycopersicum pollen tube growth. Mutants in a gene designated Defective in Induced Resistance 1-like (SpDIR1L), which encodes a small cysteine-rich protein, permitted S. lycopersicum pollen tubes to grow to the bottom third of the style. We show that SpDIR1L protein accumulation correlates with IRB strength and that species with weak or no IRBs toward S. lycopersicum pollen share a 150 bp deletion in the upstream region of SpDIR1L. These results suggest that SpDIR1L contributes to an S-RNase-independent IRB. [ABSTRACT FROM AUTHOR]

Pollen–pistil interactions are crucial for controlling plant mating. For example, S-RNase-based self-incompatibility prevents inbreeding in diverse angiosperm species. S-RNases are thought to function as specific cytotoxins that inhibit pollen that has an S-haplotype that matches one of those in the pistil. Thus, pollen and pistil factors interact to prevent mating between closely related individuals. Other pistil factors, such as HT-B, 4936-factor and the 120 kDa glycoprotein, are also required for pollen rejection but do not contribute to S-haplotype-specificity per se. Here we show that S-RNase is taken up and sorted to a vacuolar compartment in the pollen tubes. Antibodies to the 120 kDa glycoprotein label the compartment membrane. When the pistil does not express HT-B or 4936-factor, S-RNase remains sequestered, unable to cause rejection. Similarly, in wild-type pistils, compatible pollen tubes degrade HT-B and sequester S-RNase. We suggest that S-RNase trafficking and the stability of HT-B are central to S-specific pollen rejection. [ABSTRACT FROM AUTHOR]

S-RNases determine the specificity ofS-specific pollen rejection in self-incompatible plants of the Solanaceae, Rosaceae, and Scrophulariaceae. They are also implicated in at least two distinct types of unilateral interspecific incompatibility inNicotiana. However, S-RNase itself is not sufficient for most types of pollen rejection, and evidence for its direct interaction with pollen tubes is limited. Thus, non-S-RNase factors also are required for pollen rejection. As one approach to identifying such factors, we tested whether SC10-RNase fromNicotiana alatawould bind to other stylar proteinsin vitro. SC10-RNase was immobilized on Affi-gel, and binding proteins were analyzed by SDS-PAGE and immunoblotting. In addition to SC10-RNase and a small protein similar to lily chemocyanin, the most prominent binding proteins include NaTTS, 120K, and NaPELPIII, these latter three being arabinogalactan proteins previously shown to interact directly with pollen tubes. We also show that SC10-RNase and these glycoproteins migrate as a complex in a native PAGE system. Our hypothesis is that S-RNase forms a complex with these glycoproteins in the stylar ECM, that the glycoproteins interact directly with the pollen tubes and thus that the initial interaction between the pollen tube and S-RNase is indirect. [ABSTRACT FROM AUTHOR]