A pollen tube growth-promoting arabinogalactan protein from nicotiana alata is similar to the tobacco TTS protein

Upon germination on the stigma, pollen tubes elongate in the stylar transmitting tract, aided by female factors, with speed and directionality not mimicked in in vitro pollen tube growth cultures. We have shown that a stylar transmitting tissue arabinogalactan protein (AGP) from Nicotiana tabacum (tobacco), TTS protein, stimulates pollen tube growth in vivo and in vitro and attracts pollen tubes grown in a semi-in vivo culture system. It has been reported that the self-incompatible Nicotiana alata produced a stylar glycoprotein, GaRSGP, which had a backbone polypeptide that shared 97% identity with those of TTS proteins but some of its properties were different from those described for TTS proteins. We report here the characterization of a family of stylar transmitting tissue glycoproteins from N. alata that is virtually identical to tobacco TTS proteins and which we refer to as NaTTS proteins. Like their tobacco counterparts, NaTTS proteins are recognized by the traditional AGP-diagnostic reagent beta-glucosyl Yariv reagent, and they are also recognized by JIM13, a monoclonal antibody against AGP. NaTTS proteins also stimulate pollen tube elongation in vitro and attract pollen tubes in a semi-in vivo pollen tube culture system. Biochemical and immunological characterization of NaTTS proteins revealed that they have extraordinary variability in the extent of sugar modifications of their polypeptide backbones. The extent of sugar modifications on NaTTS proteins significantly affects their biochemical properties, influences how they interact with the transmitting tissue extracellular matrix, and affects their solubility from this matrix. Our results suggest that the strategy used to purify GaRSGP only recovered a less glycosylated, more tightly extracellular matrix-bound sub-population of the entire spectrum of N. alata TTS proteins.     

Stylar glycoproteins bind to S-RNase in vitro

S-RNases determine the specificity of S-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 in Nicotiana. 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 from Nicotiana alata would bind to other stylar proteins in 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.     

Expression of a Self-Incompatibility Glycoprotein (S2-Ribonuclease) from Nicotiana alata in Transgenic Nicotiana tabacum

In Nicotiana alata, self-incompatibility is controlled by a single locus, designated the S-locus, with multiple alleles. Stylar products of these alleles are ribonucleases that are secreted mainly in the transmitting tract tissues. N. tabacum plants were transformed with constructs containing the S2-cDNA and genomic S2-sequences from N. alata that were linked to the cauliflower mosaic virus 35S promoter. Unlike other genes controlled by this promoter, the genes were expressed most highly in mature floral organs. This pattern of expression was observed at both the protein and RNA levels. The S2-glycoprotein was detected in the stylar transmitting tract tissues of the transgenic plants. The transgene product was secreted, had ribonuclease activity, and was glycosylated with the correct number of glycan chains. However, the maximum level of S2-glycoprotein in styles of the transgenic plants was approximately 100-fold lower than that found in N. alata styles carrying the S2-allele. Perhaps because of this lower protein level, the plants showed no changes in the incompatibility phenotype.     

The stylar 120 kDa glycoprotein is required for S-specific pollen rejection in Nicotiana

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 x SI Nicotiana alata (S105S105 or SC10SC10) 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.     

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 extracellular 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 S_A2- and S_C10-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 S_A2- and S_C10-RNase. However, none of the chimeric S-RNases caused rejection of either S_A2- or S_C10-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.     

A lipid transfer-like protein is necessary for lily pollen tube adhesion to an in vitro stylar matrix

Flowering plants possess specialized extracellular matrices in the female organs of the flower that support pollen tube growth and sperm cell transfer along the transmitting tract of the gynoecium. Transport of the pollen tube cell and the sperm cells involves a cell adhesion and migration event in species such as lily that possess a transmitting tract epidermis in the stigma, style, and ovary. A bioassay for adhesion was used to isolate from the lily stigma/stylar exudate the components that are responsible for in vivo pollen tube adhesion. At least two stylar components are necessary for adhesion: a large molecule and a small (9 kD) protein. In combination, the two molecules induced adhesion of pollen tubes to an artificial stylar matrix in vitro. The 9-kD protein was purified, and its corresponding cDNA was cloned. This molecule shares some similarity with plant lipid transfer proteins. Immunolocalization data support its role in facilitating adhesion of pollen tubes to the stylar transmitting tract epidermis.     

Adhesion of lily pollen tubes on an artificial matrix

 We proposed that pollination in lily is a case of cell adhesion and cell movement, but experimental evidence for the adhesion event is lacking. In this study, we developed an artificial extracellular matrix that mimics the in vivo lily stylar transmitting tract. This artificial matrix was created by applying the transmitting tract exudate extracted from lily styles onto a nitrocellulose membrane. When in vitro-grown pollen tubes were applied to the matrix, they adhered by their tips to the area of the stylar exudate which is rich in arabinogalactan proteins. Once they adhered, they grew on the in vitro artificial matrix at rates faster than normal. This is the first experimental evidence demonstrating the adhesion of in vitro-grown pollen tubes, an event that has been described as common in vivo. The adhesion event is stylar exudate specific, concentration dependent, and is affected by the developmental age of the pollen tube. This bioassay for pollen tube adhesion will be used to isolate the adhesive molecules from the stylar exudate. Received: 9 December 1996 / Revision accepted: 5 May 1997     

Calcium levels increase in the lily stylar transmitting tract after pollination

Pollen tube growth in vitro requires calcium for most species but the in vivo source or reservoir of this calcium is not known. Using methods to localize calcium in situ, we confirm that low levels of calcium are detected in the transmitting tract extracellular matrix (ECM) in unpollinated lily styles. Pollination in lily induces an increase in the detectable levels of calcium in the transmitting tract ECM binding to the stylar cell and pollen tube walls. This calcium is detected in the cytoplasm and vesicles near the pollen tube tip.An erratum to this article can be found at     

Adhesion molecules in lily pollination

Adhesion occurs both between pollen tubes and between the pollen tube and transmitting tract epidermis (TTE) in lily. The stylar matrix secreted by the TTE can be isolated and used in an in vitro adhesion assay for pollen tubes. This bioassay was used to isolate two stigma/stylar adhesion molecules in lily: a pectic polysaccharide and a small cysteine-rich, basic protein we named SCA (stigma/stylar cysteine-rich adhesin). Both molecules were purified and used in an adhesion assay. Adhesion in the assay can be disrupted by treatment of the pectin with polygalacturonase and of SCA with proteinase K. The two molecules bind to each other in a pH-dependent fashion, and this binding is necessary for the adhesion assay to work. Antibodies to each of the molecules show their localization at the sites of pollen tube adhesion in the style. Pollen does not produce SCA but does bind this protein in vivo and in vitro. In vivo functional analyses are necessary to establish the roles of these molecules in lily pollination. Received: 29 October 2000 / Accepted: 17 April 2001     

A style-specific 120-kDa glycoprotein enters pollen tubes ofNicotiana alata in vivo

Pistils ofNicotiana alata (Link et Otto) contain an abundant, style-specific glycoprotein (120 kDa) that is rich in hydroxyproline and has both extensin-like and arabinogalactan-protein-like carbohydrate substituents. An antibody specific for the protein backbone of the glycoprotein was used to localise the glycoprotein in both unpollinated and pollinated pistils. The glycoprotein is evenly distributed in the extracellular matrix of the style transmitting tract of unpollinated pistils and, despite the presence of extensin-like carbohydrate substituents, is not associated with the walls of the transmitting tract cells. In pollinated pistils the 120-kDa glycoprotein is concentrated in the extracellular matrix adjacent to pollen tubes, and is also present in the cytoplasm and the cell walls of pollen tubes. Pollen tubes grown in vitro do not contain the 120-kDa glycoprotein unless it is added to the growth medium, suggesting that the 120kDa glycoprotein located in pistil-grown pollen tubes is derived from the extracellular matrix of the transmitting tract.     

A functional study of stylar hydroxyproline-rich glycoproteins during pollen tube growth

Class III pistil-specific extensin-like proteins (PELPIII) are chimeric hydroxyproline-rich glycoproteins with properties of both extensins and arabinogalactan proteins. The abundance and specific localization of PELPIII in the intercellular matrix (IM) of tobacco (Nicotiana tabacum) stylar transmitting tissue, and translocation of PELPIII from the IM into the pollen tube wall after pollination, presume the biological function of these glycoproteins to be related to plant reproduction. Here we show that in in vitro assays the translocation of PELPIII is specifically directed to the callose inner wall of the pollen tubes, indicating that protein transfer is not dependent on the physiological conditions of the transmitting tract. We designed a set of experiments to elucidate the biological function of PELPIII in the stylar IM. To study the function of the specific interaction between PELPIII proteins and the pollen tube wall, one of the PELPIII proteins (MG15) was ectopically expressed in pollen tubes and targeted to the tube wall. We also generated transgenic tobacco plants in which PELPIII proteins were silenced. In vitro bioassays were performed to test the influence of purified PELPIII on pollen tube growth, as compared to tobacco transmitting tissue-specific proteins (TTS) that were previously shown to stimulate pollen tube growth. The various tests described for activity of PELPIII proteins all gave consistent and mutually affirmative results: the biological function of PELPIII proteins is not directly related to pollen tube growth. These data show that similar stylar glycoproteins may act very differently on pollen tubes.     

A Brassica Self-Incompatibility Gene Is Expressed in the Stylar Transmitting Tissue of Transgenic Tobacco

Tobacco was transformed with a gene coding for an S-locus-specific glycoprotein of Brassica oleracea. The resulting transgenic plants showed tissue-specific and developmentally regulated expression of the introduced gene. Immunolocalization experiments showed that the Brassica gene was expressed in the stylar transmitting tissue of the transgenic plants. The pattern of expression of the introduced gene was more similar to that of the S-associated genes of Nicotiana alata than to expression in Brassica. Self-incompatibility was not conferred by the introduced gene.     

Movement of the tube cell in the lily style in the absence of the pollen grain and the spent pollen tube

Our model proposes that pollen tube growth is a form of cell movement where the tube tip can be considered analogous to a migrating cell which leaves a trail of extracellular matrix (the spent pollen tube) behind. We demonstrate that the tube cell can convey the sperm cells to the ovule and effect fertilization even in the absence of the pollen grain and the spent pollen tube. Adhesion is an integral part of cell attachment and movement in animal systems. We show that in vivo-grown pollen tubes grow beneath the cuticle of the stylar transmitting tract epidermis and directly adhere to one another and the outer wall of the epidermal cells. A fibrous wall material is found to cover the tip of the pollen tube cell wall and the surface of the transmitting tract cells where the two adhere. Fixation methods to preserve adhesive compounds were used. The pollen-tubes grown in vivo, but not in vitro, show star-shaped clusters of F-actin microfilaments in the region back from the tip, as seen by rhodamine-phalloidin staining. These configurations are similar to focal adhesions seen in moving animal cells.     

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.     

烟草柱头和花柱中阿拉伯半乳糖蛋白的定位

通过Western印迹法、免疫组织化学和超微细胞化学等技术,研究了烟草柱头和花柱中阿拉伯半乳糖蛋白（arabinogalactan-proteins,AGPs）的分布。结果表明烟草柱头和花柱组织中含有大量的AGPs,主要分布于柱头表皮细胞的细胞质和分泌层细胞的胞外基质中,且授粉前后AGPs的分布情况差异不明显;而花柱中的AGPs主要分布于表皮细胞的外层细胞壁、维管组织周围细胞的细胞质及引导组织的胞外基质中;花粉管通过后,引导组织胞外基质中AGPs减少,而花粉管细胞质和花粉管壁中检测到大量AGPs。     

NaTrxh is an essential protein for pollen rejection in Nicotiana by increasing S‐RNase activity

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, NaTrxh_SS, 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 S_C10‐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.