Pollen Recognition Research Articles

Factors Affecting Inter- and Intra-specific Pollen Rejection in Nicotiana

Abstract Inter- and intra-specific compatibility systems in the genus Nicotiana have been used to identify factors that control pollen recognition. N. alata has a classic gametophytic self-incompatibility (SI) system in which the specificity of pollen rejection is determined by a multiallelic S-locus. S-RNases are products of the S-locus, and are the factors that determine specificity in the style. In vitro mutagenesis experiments have been conducted to determine how allelic specificity is encoded in the S-RNase sequence. Plant transformation experiments have shown that S-RNases also act as factors controlling interspecific pollen rejection. By examining the effect of S-RNases on inter- and intra-specific compatibility in different genetic backgrounds, four different pollen rejection mechanisms can be recognized. S-RNases are implicated in three of these mechanisms. The dependence of pollen rejection on genetic background shows that S-RNases interact with other factors. In general, such factors can be classified in three groups based on their mode of interaction with the S-locus and other pollen–pistil interaction pathways. Some of these factors are now cloned. As more factors are cloned and characterized, it is becoming apparent that pollen–pistil interactions that were once thought to be distinct are actually interrelated.

The male determinant of self-incompatibility in Brassica.

In the S locus-controlled self-incompatibility system of Brassica, recognition of self-related pollen at the surface of stigma epidermal cells leads to inhibition of pollen tube development. The female (stigmatic) determinant of this recognition reaction is a polymorphic transmembrane receptor protein kinase encoded at the S locus. Another highly polymorphic, anther-expressed gene, SCR, also encoded at the S locus, fulfills the requirements for the hypothesized pollen determinant. Loss-of-function and gain-of-function studies prove that the SCR gene product is necessary and sufficient for determining pollen self-incompatibility specificity, possibly by acting as a ligand for the stigmatic receptor.

Cloning and Characterization of cDNA Encoding Cardosin A, an RGD-containing Plant Aspartic Proteinase

Cardosin A is an abundant aspartic proteinase from pistils of Cynara cardunculus L. whose milk-clotting activity has been exploited for the manufacture of cheese. Here we report the cloning and characterization of cardosin A cDNA. The deduced amino acid sequence contains the conserved features of plant aspartic proteinases, including the plant-specific insertion (PSI), and revealed the presence of an Arg-Gly-Asp (RGD) motif, which is known to function in cell surface receptor binding by extracellular proteins. Cardosin A mRNA was detected predominantly in young flower buds but not in mature or senescent pistils, suggesting that its expression is likely to be developmentally regulated. Procardosin A, the single chain precursor, was found associated with microsomal membranes of flower buds, whereas the active two-chain enzyme generated upon removal of PSI is soluble. This result implies a role for PSI in promoting the association of plant aspartic proteinase precursors to cell membranes. To get further insights about cardosin A, the functional relevance of the RGD motif was also investigated. A 100-kDa protein that interacts specifically with the RGD sequence was isolated from octyl glucoside pollen extracts by affinity chromatography on cardosin A-Sepharose. This result suggests that the 100-kDa protein is a cardosin A receptor and indicates that the interaction between these two proteins is apparently mediated through RGD recognition. It is possible therefore that cardosin A may have a role in adhesion-mediated proteolytic mechanisms involved in pollen recognition and growth.

Production and characterization of the Brassica oleracea self-incompatibility locus glycoprotein and receptor kinase in a baculovirus infected insect cell culture system

Self-incompatibility is a phenomenon that involves recognition of self versus non-self pollen, leading to the rejection of self-related pollen and preventing self-fertilization. In this study, we used a baculovirus-infected insect cell culture system to express two Brassica oleracea stigma-specific proteins required for self-incompatibility: the S-locus glycoprotein, a soluble cell wall-localized glycosylated protein, and the S-locus receptor kinase, a receptor-like integral plasma membrane glycoprotein with serine/threonine kinase activity. Insect cells expressing the S-locus receptor kinase were used in conjunction with immunofluorescence and a whole cell enzyme-linked immunosorbant assay to demonstrate that the receptor is targeted to the cell surface and is oriented with its N-terminal S domain towards the outside of the cell.

Signaling in pollen–pistil interactions

A complex set of cell interactions is required to achieve fertilization. The pollen grain must be recognized by the pistil, take up water, and grow a pollen tube directionally through the style in order to deliver the sperm to the ovule. In many families of flowering plants, self–fertilization can be prevented by recognition mechanisms that allow self-pollen rejection by the pistil. The self-incompatibility response is under the genetic control of a single multi–allelic locus, the (Self–incompatibility) locus. There are two major classes of self–incompatibility systems. Gametophytic self-incompatibility has been well characterized in the Solanaceae and in the Papaveraceae, while sporophytic self–incompatibility has been well characterized in the Brassicaceae. In this review article, we present recent advances in understanding the signals mediating pollen recognition and pollen tube growth, in both compatible and incompatible interactions.

The mating game: pollination and fertilization in flowering plants

Recent work has revealed signaling molecules that control pollination, including small peptides that mediate pollen recognition and glycoproteins that support pollen tube growth. The polarized growth of pollen tubes requires a calcium-mediated signal cascade, and cues derived from the haploid and diploid ovule cells guide pollen tubes to the eggs.

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.

Identification of Residues in a Hydrophilic Loop of the Papaver rhoeas S Protein That Play a Crucial Role in Recognition of Incompatible Pollen

Identification of Residues in a Hydrophilic Loop of the Papaver rhoeas S Protein That Play a Crucial Role in Recognition of Incompatible Pollen

Are the Hypervariable Regions of S RNases Sufficient for Allele-Specific Recognition of Pollen?

In a recent research article published in THE PLANT CELL (“Hypervariable Domains of Self-Incompatibility RNases Mediate Allele-Specific Pollen Recognition”), Matton et al. ([1997][1]) show that the pollen recognition function of the Solanum chacoense S11 RNase can be converted to that of the S13

Are the Hypervariable Regions of S RNases Sufficient for Allele-Specific Recognition of Pollen? [with Reply

Are the Hypervariable Regions of S RNases Sufficient for Allele-Specific Recognition of Pollen? [with Reply

Self-incompatibility and other pollen-pistil interactions

Self-incompatibility allows plants to recognize and rejectpollen from the same plant, thereby reducing inbreeding. Although in most cases self-incompatibility is controlled by a single genetic locus, recent results show that surprisingly complex signal transduction pathways and many players are involved in pollen recognition and rejection.

Hypervariable Domains of Self-Incompatibility RNases Mediate Allele-Specific Pollen Recognition.

Self-incompatibility (SI) in angiosperms is a genetic mechanism that promotes outcrossing through rejection of self-pollen. In the Solanaceae, SI is determined by a multiallelic S locus whose only known product is an S RNase. S RNases show a characteristic pattern of five conserved and two hypervariable regions. These are thought to be involved in the catalytic function and in allelic specificity, respectively. When the Solanum chacoense S12S14 genotype is transformed with an S11 RNase, the styles of plants expressing significant levels of the transgene reject S11 pollen. A previously characterized S RNase, S13, differs from the S11 RNase by only 10 amino acids, four of which are located in the hypervariable regions. When S12S14 plants were transformed with a chimeric S11 gene in which these four residues were substituted with those present in the S13 RNase, the transgenic plants acquired the S13 phenotype. This result demonstrates that the S RNase hypervariable regions control allelic specificity.

Hypervariable Domains of Self-Incompatibility RNases Mediate Allele-Specific Pollen Recognition

Hypervariable Domains of Self-Incompatibility RNases Mediate Allele-Specific Pollen Recognition

Exchanging sequence domains between S-RNases from Nicotiana alata disrupts pollen recognition.

In self-incompatible plants of the Solanaceae, the specificity of pollen rejection is controlled by a single multiallelic S-locus. Pollen tube growth is inhibited in the style when its single S-allele matches either S-allele present in the diploid pistil. Each S-allele encodes an S-RNase with a unique sequence. S-RNases are secreted into the extra-cellular matrix of the transmitting tract which guides pollen tubes toward the ovary. Although it is known that S-RNases are the determinants of S-allele specificity in the pistil, it is not known how allele-specific information is encoded in the sequence. Therefore, we exchanged domains between S-RNases with different recognition specificities and expressed the chimeric proteins in transgenic plants to determine their effects on pollination behavior. Nine chimeric constructs were prepared in which domains from Nicotiana alata SA2- and SC10-RNases were exchanged. Among these nine constructs, the entire S-RNase sequence was sampled by exchanging single variable domains as well as larger blocks of contiguous sequences. The chimeric S-RNases retained enzymatic activity and were expressed at levels comparable to control transformants expressing SA2- and SC10-RNases. However, none of the chimeric S-RNases caused rejection of either SA2- or SC10-pollen. We conclude that the recognition function of S-RNases can be disrupted by alterations in many parts of the sequence. It appears that the recognition function of S-RNase is not localized to a specific domain.

Blazing New Trails (Pollen Tube Guidance in Flowering Plants).

Higher plants minimize their exposure to pollen from diverse species by precise regulation of flowering time, development of floral organs, and interactions with specific pollinators. These measures do not guarantee success, however, because of the vast quantities of pollen in the environment. Consequently, to ensure that only the appropriate sperm are delivered to the eggs, many plants have evolved elaborate signaling pathways that monitor the pollen as it grows into the interior of the pistil. Signals are exchanged at every step along this pathway, beginning with the recognition of pollen by cells on the pistil surface and continuing as the pollen grains extend polarized projections (pollen tubes) that invade the pistil and migrate to the eggs. As the pollen tubes race toward their targets, these signals guide the deposition of new membrane and cell wall components and the transportation of sperm and other cellular contents to the tip of the growing tube. Whether the signals that direct pollen tube growth come from female tissues or from the pollen tube itself remains unknown; successful navigation most likely depends on the interplay of both male and female signals. These signals could be structural or chemical in nature, and probably comprise a combination of positive cues that guide pollen tubes to the eggs and negative signals that repel other tubes. Recent investigations into pollen tube guidance have brought the answers to some of these questions within reach.

The self-incompatibility (S) haplotypes of Brassica contain highly divergent and rearranged sequences of ancient origin.

In Brassica, the recognition of self-related pollen by the stigma is controlled by the highly polymorphic S locus that encodes several linked and coadapted genes and can span several hundred kilobases. We used pulsed-field gel electrophoresis to analyze the structure of different S haplotypes. We show that the S2 and S13 haplotypes of Brassica oleracea contain extensive sequence divergence and rearrangement relative to each other. In contrast, haplotypic configuration is more conserved between B. oleracea S13 and B. campestris S8, two haplotypes that have been proposed to be derived from a common ancestral haplotype based on sequence comparisons. These results support the view that extensive restructuring of the S locus preceded speciation in Brassica. This structural heteromorphism, together with haplotype-specific sequences, may suppress recombination within the S locus complex, potentially providing a mechanism for maintaining the linkage of coadapted allelic combinations of genes over time.

Identification of genes required for pollen‐stigma recognition in Arabidopsis thaliana

In higher plants, cell-cell recognition reactions taking place following pollination allow the selective restriction of self-pollination and/or interspecific pollination. Many of these systems function by regulating the process of water transfer from the cells found at the stigmatic surface to the individual pollen grain. Interspecific pollination studies on the cruciferous weed Arabidopsis thaliana revealed only a broad specificity of pollen recognition such that pollen from all tested members of the crucifer family were recognized, whereas pollen from almost all other species failed to hydrate. Genetic analysis of A. thaliana has identified three genes that are essential for this recognition process. Recessive mutations in any of these genes result in male sterility due to the production of pollen grains that fail to hydrate when placed on the stigma, but that are capable of hydrating and growing a pollen tube in vitro. Results from mixed pollination experiments suggest that the mutant pollen grains specifically lack a functional pollen-stigma recognition system. All three mutations described also result in a defect in the wax layer normally found on stems and leaves, similar to previously described eceriferum (cer) mutations. Genetic complementation and mapping experiments demonstrated that the newly identified mutants are allelic to the previously identified genes cer1, cer3 and cer6. TEM analysis of the ultrastructure of the pollen coating revealed that all of the mutant pollen grains bear coatings of normal thickness and that tryphine lipid droplets are missing in cer1-147, are reduced in size in cer6-2654 and appear normal in cer3-2186.(ABSTRACT TRUNCATED AT 250 WORDS)

An anther-specific gene encoded by an S locus haplotype of Brassica produces complementary and differentially regulated transcripts.

The self-incompatibility locus of Brassica consists of a coadapted gene complex that contains at least two genes required for the recognition and inhibition of pollen by the stigma when self-pollinated. Here, we report the identification of a third S locus-linked gene from the S2 haplotype of Brassica oleracea. This gene, which we designated SLA (for S Locus Anther), is a novel gene with an unusual structure. SLA is transcribed from two promoters to produce two complementary anther-specific transcripts, one spliced and the other unspliced, that accumulate in an antiparallel manner in developing microspores and anthers. The sequence of the spliced transcript showed the presence of two open reading frames that predict proteins of 10 and 7.5 kD. Neither transcript was produced in a self-compatible B. napus strain carrying an S2-like haplotype, indicating that the SLA gene in this strain is nonfunctional. Interestingly, sequences related to SLA were not detected in DNA or RNA from plants carrying S haplotypes other than S2. The haplotype specificity of SLA, its anther-specific expression, and its physical linkage to the S locus are properties expected for a gene that encodes a determinant of S2 specificity in pollen.

Breakdown of Self-incompatibility in Brassica by the Antisense RNA of the SLG Gene.

The self incompatibility (SI) system of Brassica is sporophytically controlled by multiallenic genes at an S-locus. Two genes, SLG (S-locus glycoprotein) and SRK(S-receptor protein kinase), on the S-locus are thought to play an important role in recognition of the self pollen, though there is no direct evidence. We introduced the antisense SLG gene to self incompatible Brassica species through Agrobacterium-mediated transformation. We found that a transgenic plant of B. raga (syn. B. campestris) could set seeds after self pollination, and that no SLG protein was detectable in a gel blot analysis of the transgenic plant. These observations provide strong evidence that SLG and/or SRK are directly involved in the pollen-stigma recognition of SI.

S-RNase expressed in transgenic Nicotiana causes S-allele-specific pollen rejection.

Many angiosperms employ self-incompatibility systems to prevent inbreeding. The simple genetics of such systems have made them attractive models of plant cellular communication. Implicit in the single locus genetics is that only one or a few gene products are necessary for recognition and rejection of incompatible pollen. Results in the sporophytic system of the Brassicaceae suggest that different S-locus products are responsible for the pollen and pistil parts of the recognition and rejection response. In solanaeceous plants, which have a gametophytic self-incompatibility system, the S locus product responsible for the pollen portion of the interaction has not been identified, but ribonucleases encoded by the S-locus (S-RNases) are strongly implicated in the style part of the recognition and rejection reaction. In Nicotiana alata, pollen recognition and rejection occur if its S-allele matches either S-allele in the style. The putative stylar S-RNase is abundant in the transmitting tract, and pollen rejection may be related to action of S-RNase on pollen RNAs. Efforts to understand the molecular basis for pollen recognition and rejection have been limited by the lack of a system for manipulating and expressing S-RNases. Here we use the promoter of a style-expressed gene from tomato to obtain high levels of S-RNase expression in transgenic Nicotiana. Recognition and rejection of N. alata pollen S-alleles occur faithfully in the transgenic plants. Our results show that S-RNases alone are sufficient for pollen rejection in this system.