Gametophytic self-incompatibility: contrasting mechanisms for Nicotiana and Papaver

Since Darwin first noted that not all plants produce self-seed, several mechanisms that regulate the acceptance or rejection of pollen during fertilization have been recognized, of which self-incompatibility (SI) is the most widespread. Over the past few years much progress has been made in understanding the molecular and cellular processes involved in SI. Here we review recent studies of the SI systems of Nicotiana alata and Papaver rhoeas. The SI systems are both determined by a single, multi-allelic gametophytically controlled S-gene, but involve quite different mechanisms.     

Alterations in the actin cytoskeleton of pollen tubes are induced by the self-incompatibility reaction in Papaver rhoeas

Self-incompatibility (SI) is a genetically controlled process used to prevent self-pollination. In Papaver rhoeas, the induction of SI is triggered by a Ca(2)+-dependent signaling pathway that results in the rapid and S allele-specific inhibition of pollen tube tip growth. Tip growth of cells is dependent on a functioning actin cytoskeleton. We have investigated the effect of self-incompatibility (S) proteins on the actin cytoskeleton in poppy pollen tubes. Here, we report that the actin cytoskeleton of incompatible pollen tubes is rapidly and dramatically rearranged during the SI response, not only in our in vitro SI system but also in vivo. We demonstrate that nonspecific inhibition of growth does not result in similar actin rearrangements. Because the SI-induced alterations are not observed if growth stops, this clearly demonstrates that these alterations are triggered by the SI signaling cascade rather than merely resulting from the consequent inhibition of growth. We establish a detailed time course of events and discuss the mechanisms that might be involved. Our data strongly implicate a role for the actin cytoskeleton as a target for signaling pathways involved in the SI response of P. rhoeas.     

RNaseI from Escherichia coli cannot substitute for S-RNase in rejection of Nicotiana plumbaginifolia pollen

Unilateral incompatibility often occurs between self-incompatible (SI) species and their self-compatible (SC) relatives. For example, SI Nicotiana alata rejects pollen from SC N. plumbaginifolia, but the reciprocal pollination is compatible. This interspecific pollen rejection system closely resembles intraspecific S-allele-specific pollen rejection. However, the two systems differ in degree of specificity. In SI, rejection is S-allele-specific, meaning that only a single S-RNase causes rejection of pollen with a specific S genotype. Rejection of N. plumbaginifolia pollen is less specific, occurring in response to almost any S-RNase. Here, we have tested whether a non-S-RNase can cause rejection of N. plumbaginifolia pollen. The Escherichia coli rna gene encoding RNaseI was engineered for expression in transgenic (N. plumbaginifolia × SC N. alata) hybrids. Expression levels and pollination behavior of hybrids expressing E. coli RNaseI were compared to controls expressing S_A2-RNase from N. alata. Immunoblot analysis and RNase activity assays showed that RNaseI and S_A2-RNase were expressed at comparable levels. However, expression of S_A2-RNase caused rejection of N. plumbaginifolia pollen, whereas expression of RNaseI did not. Thus, in this system, RNase activity alone is not sufficient for rejection of N. plumbaginifolia pollen. The results suggest that S-RNases may be specially adapted to function in pollen rejection.     

Actin depolymerization is sufficient to induce programmed cell death in self-incompatible pollen

Self-incompatibility (SI) prevents inbreeding through specific recognition and rejection of incompatible pollen. In incompatible Papaver rhoeas pollen, SI triggers a Ca2+ signaling cascade, resulting in the inhibition of tip growth, actin depolymerization, and programmed cell death (PCD). We investigated whether actin dynamics were implicated in regulating PCD. Using the actin-stabilizing and depolymerizing drugs jasplakinolide (Jasp) and latrunculin B, we demonstrate that changes in actin filament levels or dynamics play a functional role in initiating PCD in P. rhoeas pollen, triggering a caspase-3-like activity. Significantly, SI-induced PCD in incompatible pollen was alleviated by pretreatment with Jasp. This represents the first account of a specific causal link between actin polymerization status and initiation of PCD in a plant cell and significantly advances our understanding of the mechanisms involved in SI.     

S RNase and Interspecific Pollen Rejection in the Genus Nicotiana: Multiple Pollen-Rejection Pathways Contribute to Unilateral Incompatibility between Self-Incompatible and Self-Compatible Species

In self-incompatible (SI) plants, the S locus acts to prevent growth of self-pollen and thus promotes outcrossing within the species. Interspecific crosses between SI and self-compatible (SC) species often show unilateral incompatibility that follows the SI x SC rule: SI species reject pollen from SC species, but the reciprocal crosses are usually compatible. The general validity of the SI x SC rule suggests a link between SI and interspecific pollen rejection; however, this link has been questioned because of a number of exceptions to the rule. To clarify the role of the S locus in interspecific pollen rejection, we transformed several Nicotiana species and hybrids with genes encoding SA2 or SC10 RNase from SI N. alata. Compatibility phenotypes in the transgenic plants were tested using pollen from three SC species showing unilateral incompatibility with N. alata. S RNase was implicated in rejecting pollen from all three species. Rejection of N. plumbaginifolia pollen was similar to S allele-specific pollen rejection, showing a requirement for both S RNase and other genetic factors from N. alata. In contrast, S RNase-dependent rejection of N. glutinosa and N. tabacum pollen proceeded without these additional factors. N. alata also rejects pollen from the latter two species through an S RNase-independent mechanism. Our results implicate the S locus in all three systems, but it is clear that multiple mechanisms contribute to interspecific pollen rejection.     

Ca2+-independent phosphorylation of a 68 kDa pollen protein is stimulated by the self-incompatibility response in Papaver rhoeas

The self-incompatibility (SI) response in Papaver rhoeas involves a Ca²⁺-based signalling pathway, which mediates the SI-specific inhibition of incompatible pollen. We have previously reported the identification of p26.1, a pollen protein whose phosphorylation was increased specifically as a consequence of the SI response. We have investigated whether further specific protein phosphorylation events are induced in P. rhoeas pollen. Here we report the identification of an additional pollen protein, p68, which also responds to S proteins by an increase in its phosphorylation state. This phosphorylation event occurs in living pollen tubes grown in vitro , and can be observed specifically when pollen is challenged with biologically active S proteins that are incompatible with the S alleles carried by the pollen and not when pollen was challenged with compatible S proteins. The timing of the increase in phosphorylation of p68 is temporally later than that of p26.1, occurring between 240 sec and 400 sec after challenge. This suggests that its phosphorylation is downstream of p26.1 in the SI signalling pathway(s). Surprisingly, the kinases responsible for the phosphorylation of p68 are not Ca²⁺-dependent. This, and the later timing of the p68 response, suggests that a 'second wave' of Ca²⁺-independent signalling may follow the initial Ca²⁺-dependent SI signalling. This indicates that the SI signalling pathway(s) in pollen may be quite complex.     

Evidence for DNA fragmentation triggered in the self-incompatibility response in pollen of Papaver rhoeas

Studies of the molecular and biochemical basis of self-incompatibility (SI) in Papaver rhoeas have revealed much about the signalling pathways triggered in pollen early in this response. The aim of the current investigation was to begin to study downstream events in order to elucidate some of the later cellular responses involved in the SI response and identification of the mechanisms controlling the irreversible inhibition of pollen tube growth. We have used the FragEL assay to investigate if there is any evidence for DNA fragmentation stimulated in pollen of P. rhoeas in an S-specific manner. Our data clearly demonstrate that S proteins are responsible for triggering this, specifically in incompatible, and not compatible, pollen. DNA fragmentation was first detected in incompatible pollen tubes 4 h after challenge with S proteins, and continued to increase for a further 10 h. This provides the first evidence, to our knowledge, that this phenomenon is associated with the SI response. We also demonstrate that mastoparan, which increases [Ca2+]i, also triggers DNA fragmentation in these pollen tubes, thereby implicating an involvement of Ca2+ signalling in this process. Together, our data represent a significant breakthrough in understanding of the SI response in Papaver pollen.     

Expression of 10 S-class SLF-like genes in Nicotiana alata pollen and its implications for understanding the pollen factor of the S locus

The S locus of Nicotiana alata encodes a polymorphic series of ribonucleases (S-RNases) that determine the self-incompatibility (SI) phenotype of the style. The pollen product of the S locus (pollen S) in N. alata is unknown, but in species from the related genus Petunia and in self-incompatible members of the Plantaginaceae and Rosaceae, this function has been assigned to an F-box protein known as SLF or SFB. Here we describe the identification of 10 genes (designated DD1-10) encoding SLF-related proteins that are expressed in N. alata pollen. Because our approach to cloning the DD genes was based on sequences of SLFs from other species, we presume that one of the DD genes encodes the N. alata SLF ortholog. Seven of the DD genes were exclusively expressed in pollen and a low level of sequence variation was found in alleles of each DD gene. Mapping studies confirmed that all 10 DD genes were linked to the S locus and that at least three were located in the same chromosomal segment as pollen S. Finally, the different topologies of the phylogenetic trees produced using available SLF-related sequences and those produced using S-RNase sequences suggests that pollen S and the S-RNase have different evolutionary histories.     

Increased Phosphorylation of a 26-kD Pollen Protein Is Induced by the Self-Incompatibility Response in Papaver rhoeas

We have investigated whether specific protein phosphorylation events are induced in Papaver rhoeas pollen as a consequence of the self-incompatibility (SI) response. Pollen grown in vitro in the presence of 32P-orthophosphate was challenged with biologically active recombinant S proteins, and pollen proteins were extracted and analyzed. The results provide strong evidence that the increased phosphorylation of a 26-kD protein of pl 6.2, p26, is specifically induced by the SI response. This phosphorylation event occurs in living pollen tubes and was observed specifically when pollen was challenged with S proteins that are incompatible with the S alleles carried by the pollen and not when pollen was challenged with compatible or incompatible heat-denatured S proteins. Further characterization demonstrated that p26 comprises two phosphoproteins, p26.1 and p26.2, that are found in soluble and microsomal fractions, respectively. Increased phosphorylation of p26.1 is implicated in the SI response and appears to be Ca2+ and calmodulin dependent. These data argue for the involvement of a Ca2+-dependent protein kinase requiring calmodulin-like domains, whose activation comprises an intracellular signal mediating the SI response in P. rhoeas pollen.     

高等植物自交不亲和反应中花粉管信号传导研究进展(综述)

高等植物自交不亲和反应是由基因控制、避免发生自花授粉的一种机制。本文介绍以虞美人为主的高等植物在自交不亲和反应中肌动蛋白骨架的动态变化及Ca2 的时空变化,着重阐述花粉管生长被抑制的最初信号传导。     

Ribonuclease activity of Petunia inflata S proteins is essential for rejection of self-pollen.

S proteins, pistil-specific ribonucleases that cosegregate with S alleles, have previously been shown to control rejection of self-pollen in Petunia inflata and Nicotiana alata, two solanaceous species that display gametophytic self-incompatibility. The ribonuclease activity of S proteins was thought to degrade RNA of self-pollen tubes, resulting in the arrest of their growth in the style. However, to date no direct evidence has been obtained. Here, the ribonuclease activity of S3 protein of P. inflata was abolished, and the effect on the pistil's ability to reject S3 pollen was examined. The S3 gene was mutagenized by replacing the codon for His-93, which has been implicated in ribonuclease activity, with a codon for asparagine, and the mutant S3 gene was introduced into P. inflata plants of S1S2 genotype. Two transgenic plants produced a level of mutant S3 protein comparable to that of the S3 protein produced in self-incompatible S1S3 and S2S3 plants, yet they failed to reject S3 pollen. The mutant S3 protein produced in these two transgenic plants did not exhibit any detectable ribonuclease activity. We have previously shown that transgenic plants (S1S2 plants transformed with the wild-type S3 gene) producing a normal level of wild-type S3 protein acquired the ability to reject S3 pollen completely. Thus, the results reported here provide direct evidence that the biochemical mechanism of gametophytic self-incompatibility in P. inflata involves the ribonuclease activity of S proteins.     

Identification of residues in a hydrophilic loop of the Papaver rhoeas S protein that play a crucial role in recognition of incompatible pollen.

The self-incompatibility response involves S allele-specific recognition between stigmatic S proteins and incompatible pollen. This response results in pollen inhibition. Defining the amino acid residues within the stigmatic S proteins that participate in S allele-specific inhibition of incompatible pollen is essential for the elucidation of the molecular basis of the self-incompatibility response. We have constructed mutant derivatives of the S1 protein from Papaver rhoeas by using site-directed mutagenesis and have tested their biological activity. This has enabled us to identify amino acid residues in the stigmatic S proteins of P. rhoeas that are required for S-specific inhibition of incompatible pollen. We report here the identification of several amino acid residues in the predicted hydrophilic loop 6 of the P. rhoeas stigmatic S1 protein that are involved in the inhibition of S1 pollen. Mutation of the only hypervariable amino acid, which is situated in this loop, resulted in the complete loss of ability of the S protein to inhibit S1 pollen. This clearly demonstrates that this residue plays a crucial role in pollen recognition and may also participate in defining allelic specificity. We have also established the importance of highly conserved amino acids adjacent to this hypervariable site. Our studies demonstrate that both variable and conserved amino acids in the region of the S protein corresponding to surface loop 6 are key elements that play a role in the recognition and inhibition of incompatible pollen in the pollen-pistil self-incompatibility reaction.     

Cytomechanical properties of papaver pollen tubes are altered after self-incompatibility challenge

Self-incompatibility (SI) in Papaver rhoeas triggers a ligand-mediated signal transduction cascade, resulting in the inhibition of incompatible pollen tube growth. Using a cytomechanical approach we have demonstrated that dramatic changes to the mechanical properties of incompatible pollen tubes are stimulated by SI induction. Microindentation revealed that SI resulted in a reduction of cellular stiffness and an increase in cytoplasmic viscosity. Whereas the former cellular response is likely to be the result of a drop in cellular turgor, we hypothesize that the latter is caused by as yet unidentified cross-linking events. F-actin rearrangements, a characteristic phenomenon for SI challenge in Papaver, displayed a spatiotemporal gradient along the pollen tube; this suggests that signal propagation occurs in a basipetal direction. However, unexpectedly, local application of SI inducing S-protein did not reveal any evidence for localized signal perception in the apical or subapical regions of the pollen tube. To our knowledge this represents the first mechanospatial approach to study signal propagation and cellular responses in a well-characterized plant cell system. Our data provide the first evidence for mechanical changes induced in the cytoplasm of a plant cell stimulated by a defined ligand.     

Genomic organization of the Papaver rhoeas self-incompatibility S(1) locus

The self-incompatibility (SI) response in Papaver rhoeas depends upon the cognate interaction between a pollen-expressed receptor and a stigmatically expressed ligand. The genes encoding these components are situated within the S-locus. In order for SI to be maintained, the genes encoded by the S-locus must be co-inherited with no recombination between them. Several hypotheses, including sequence heterogeneity and chromosomal position, have been put forward to explain the maintenance of the S-locus in the SI systems of the Brassicaceae and the Solanaceae. A region of the Papaver rhoeas genome encompassing part of the self-incompatibility S(1) locus has been cloned and sequenced. The clone contains the gene encoding the stigmatic component of the response, but does not contain a putative pollen S-gene. The sequence surrounding the S(1) gene contains several diverse repetitive DNA elements. As such, the P. rhoeas S-locus bears similarities to the S-loci of other SI systems. An attempt to localize the P. rhoeas S-locus using fluorescence in situ hybridization (FISH) has also been made. The potential relevance of the findings to mechanisms of recombination suppression is discussed.     

Growth of Pollen Tubes of Papaver rhoeas Is Regulated by a Slow-Moving Calcium Wave Propagated by Inositol 1,4,5-Trisphosphate

A signaling role for cytosolic free Ca2+ ([Ca2+]i) in regulating Papaver rhoeas pollen tube growth during the self-incompatibility response has been demonstrated previously. In this article, we investigate the involvement of the phosphoinositide signal transduction pathway in Ca2+-mediated pollen tube inhibition. We demonstrate that P. rhoeas pollen tubes have a Ca2+-dependent polyphosphoinositide-specific phospholipase C activity that is inhibited by neomycin. [Ca2+]i imaging after photolysis of caged inositol (1,4,5)-trisphosphate (Ins[1,4,5]P3) in pollen tubes demonstrated that Ins(1,4,5)P3 could induce Ca2+ release, which was inhibited by heparin and neomycin. Mastoparan, which stimulated Ins(1,4,5)P3 production, also induced a rapid increase in Ca2+, which was inhibited by neomycin. These data provide direct evidence for the involvement of a functional phosphoinositide signal-transducing system in the regulation of pollen tube growth. We suggest that the observed Ca2+ increases are mediated, at least in part, by Ins(1,4,5)P3-induced Ca2+ release. Furthermore, we provide data suggesting that Ca2+ waves, which have not previously been reported in plant cells, can be induced in pollen tubes.     

The self-incompatibility response in Papaver rhoeas is mediated by cytosolic free calcium

The role of Ca²⁺ signalling during the self-incompatibility (SI) response in Papaver rhoeas L. has been investigated using Ca²⁺-sensitive dyes. Pollen tubes were micro-injected with Calcium Green-1 and cytosolic free calcium ([Ca²⁺]_i) imaged using laser scanning confocal microscopy (LSCM). Addition of incompatible stigmatic S-glycoproteins induced a transient increase in the level of [Ca²⁺]_i in pollen tubes. In contrast, no rise in [Ca²⁺]_i was detectable after addition of either compatible or heat-denatured incompatible stigmatic S-glycoproteins. The elevation of [Ca²⁺]_i was followed by the specific inhibition of pollen tube growth in incompatible reactions. It has been shown previously that gene expression in pollen tubes is switched on during an incompatible reaction. Since the [Ca²⁺]_i transient appeared to originate from the region where the nuclei are located, Ca²⁺ may be involved in locally regulating the expression of these genes. The photoactivation of caged Ca²⁺ to artificially elevate [Ca²⁺]_i resulted in the inhibition of pollen tube growth and thus mimicked the SI response. Taken together, the results provide an important link between a transient rise in [Ca²⁺]_i and the biological phenomenon of inhibition of pollen tube growth and demonstrate, for the first time, direct evidence that the SI response in P. rhoeas is mediated by [Ca²⁺]_i.     

Self-incompatibility in Papaver: signalling to trigger PCD in incompatible pollen

Sexual reproduction in higher plants uses pollination, involving interactions between pollen and pistil. Self-incompatibility (SI) prevents self-fertilization, providing an important mechanism to promote outbreeding. SI is controlled by the S-locus; discrimination occurs between incompatible pollen, which is rejected, while compatible pollen can achieve fertilization. In Papaver rhoeas, S proteins encoded by the pistil part of the S-locus interact with incompatible pollen to effect rapid inhibition of tip growth. This self-incompatible interaction triggers a Ca(2+)-dependent signalling cascade. SI-specific events triggered in incompatible pollen include rapid depolymerization of the actin cytoskeleton; phosphorylation of soluble inorganic pyrophosphatases, and activation of a MAPK. It has recently been shown that programmed cell death (PCD) is triggered by SI. This provides a precise mechanism for the specific destruction of 'self' pollen. Recent data providing evidence for SI-induced caspase-3-like protease activity, and the involvement of actin depolymerization and MAPK activation in SI-mediated PCD will be discussed. These studies not only significantly advance our understanding of the mechanisms involved in SI, but also contribute to our understanding of functional links between signalling components and initiation of PCD in a plant cell. Recent data demonstrating SI-mediated modification of soluble inorganic pyrophosphatases are also described.     

Pollination in Nicotiana alata stimulates synthesis and transfer to the stigmatic surface of NaStEP, a vacuolar Kunitz proteinase inhibitor homologue

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.