Incompatible Pollen Tube Growth Research Articles

The influence of the pollination compatibility type on the pistil S-RNase expression in European pear (Pyrus communis).

The S-RNase gene plays an essential role in the gametophytic self-incompatibility (GSI) system of Pyrus. It codes for the stylar-expressed S-RNase protein which inhibits the growth of incompatible pollen tubes through cytotoxicity and the induction of programmed cell death in the pollen tube. While research on the Pyrus GSI system has primarily focused on the S-RNase gene, there is still a lack of insight into its spatiotemporal expression profile and the factors that regulate it. Previous studies have suggested that S-RNase expression in the style is influenced by pollination and is dependent on the compatibility type. We here continue on this basic hypothesis by analyzing the spatiotemporal expression of the S-RNase alleles in Pyrus communis "Conference" styles in response to different types of pollination; namely, upon full- and semi-compatible pollination and upon incompatible selfing. The results revealed that temporal dynamics of S-RNase expression are influenced by the pollen's compatibility type, indicating the presence of a signaling mechanism between pollen and style to control S-RNase production during pollen tube growth. In our experiment, S-RNase expression continuously decreased after cross-pollination and in the unpollinated control. However, after a fully incompatible pollination, S-RNase expression remained constant. Finally, semi-compatible pollination showed a initially constant S-RNase expression for both alleles followed by a strong decrease in expression. Based on these results and previous findings, we propose a regulatory mechanism to explain the effect of pollination and the associated compatibility type on S-RNase expression in the style. This proposed mechanism could be used as a starting point for future research.

PbrBZR1 interacts with PbrARI2.3 to mediate brassinosteroid-regulated pollen tube growth during self-incompatibility signaling in pear.

S-RNase-mediated self-incompatibility (SI) prevents self-fertilization and promotes outbreeding to ensure genetic diversity in many flowering plants, including pear (Pyrus sp.). Brassinosteroids (BRs) have well-documented functions in cell elongation, but their molecular mechanisms in pollen tube growth, especially in the SI response, remain elusive. Here, exogenously applied brassinolide (BL), an active BR, countered incompatible pollen tube growth inhibition during the SI response in pear. Antisense repression of BRASSINAZOLE-RESISTANT1 (PbrBZR1), a critical component of BR signaling, blocked the positive effect of BL on pollen tube elongation. Further analyses revealed that PbrBZR1 binds to the promoter of EXPANSIN-LIKE A3 (PbrEXLA3) to activate its expression. PbrEXLA3 encodes an expansin that promotes pollen tube elongation in pear. The stability of dephosphorylated PbrBZR1 was substantially reduced in incompatible pollen tubes, where it is targeted by ARIADNE2.3 (PbrARI2.3), an E3 ubiquitin ligase that is strongly expressed in pollen. Our results show that during the SI response, PbrARI2.3 accumulates and negatively regulates pollen tube growth by accelerating the degradation of PbrBZR1 via the 26S proteasome pathway. Together, our results show that an ubiquitin-mediated modification participates in BR signaling in pollen and reveal the molecular mechanism by which BRs regulate S-RNase-based SI.

Research Progress on Calcium Ion in Gametophytic Self-Incompatibility

Abstract Calcium ions are involved in plant self-incompatibility response as important signaling substances in cells. In the sporophytic self-incompatibility response, Ca2+ enters the stigma papilla cells and plays a key role in inhibiting incompatible pollen tube growth. In the gametophytic self-incompatibility reaction of Papaveraceae, the female determinants in the style (PrsS) and the male determinants in the pollen (PrpS) recognize each other, promote extracellular Ca2+ influx into the incompatible pollen tube, destroy the calcium ion gradient at the tip of the pollen tube, and inhibit the pollen tube growth. In the S-RNase-based Rosaceae game-tophytic self-incompatibility response, it is still unclear how the S-RNase interacts with the male determinant and how the S-RNase specifically degrades the RNA in the pollen tube. Therefore, we reviewed the research progress on the role of Ca2+ in self-incompatibility and, based on our research results, proposed a role model of Ca2+ as a signal substance in the gametophyte self-incompatibility response in Rosaceae.

Exploration of molecular mechanism of intraspecific cross-incompatibility in sweetpotato by transcriptome and metabolome analysis.

Cross-incompatibility, frequently happening in intraspecific varieties, has seriously restricted sweetpotato breeding. However, the mechanism of sweetpotato intraspecific cross-incompatibility (ICI) remains largely unexplored, especially for molecular mechanism. Treatment by inducible reagent developed by our lab provides a method to generate material for mechanism study, which could promote incompatible pollen germination and tube growth in the ICI group. Based on the differential phenotypes between treated and untreated samples, transcriptome and metabolome were employed to explore the molecular mechanism of sweetpotato ICI in this study, taking varieties ‘Guangshu 146’ and ‘Shangshu 19’, a typical incompatible combination, as materials. The results from transcriptome analysis showed oxidation–reduction, cell wall metabolism, plant–pathogen interaction, and plant hormone signal transduction were the essential pathways for sweetpotato ICI regulation. The differentially expressed genes (DEGs) enriched in these pathways were the important candidate genes to response ICI. Metabolome analysis showed that multiple differential metabolites (DMs) involved oxidation–reduction were identified. The most significant DM identified in comparison between compatible and incompatible samples was vitexin-2-O-glucoside, a flavonoid metabolite. Corresponding to it, cytochrome P450s were the most DEGs identified in oxidation–reduction, which were implicated in flavonoid biosynthesis. It further suggested oxidation–reduction play an important role in sweetpotato ICI regulation. To validate function of oxidation–reduction, reactive oxygen species (ROS) was detected in compatible and incompatible samples. The green fluorescence was observed in incompatible but not in compatible samples. It indicated ROS regulated by oxidation–reduction is important pathway to response sweetpotato ICI. The results in this study would provide valuable insights into molecular mechanisms for sweetpotato ICI.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11103-022-01259-8.

Transcriptomic analysis identifies critical signaling components involved in the self-incompatibility response in Chinese cabbage

Self-incompatibility (SI) is a genetic mechanism in plants to prevent inbreeding, promote outcrossing and thereby increase genetic diversity. Although current studies have well revealed the pathway of SI recognition specificity underlying interaction of female and male determinants in Brassica, the inhibition mechanism of incompatible pollen germination and tube growth is still deficient. In this study, histological analysis showed that the pollen germination and tube growth were obviously suppressed after self-pollination in self-incompatible Chinese cabbage. Furthermore, RNA-seq and gene expression analysis were performed to identify the differentially expressed genes (DEGs) involved in SI response of Chinese cabbage. A total of 59 DEGs belonging to known SI signaling factors and Ca2+-binding proteins were identified in Chinese cabbage after incompatible self-pollination. QRT-PCR analysis found that several genes were significantly differentially expressed during SI response. Chromosomal localization and potential duplicated genes were also predicted. Additionally, spatial-temporal expression profiling revealed that 15 DEGs were specifically expressed in the floral organ, some of which exhibited higher expressions in the anther and style. These findings will provide insights into the functional exploration of SI signaling factors in Chinese cabbage, and promote the further understanding of SI signaling networks in Brassica.

PELPIII: the class III pistil‐specific extensin‐like Nicotiana tabacum proteins are essential for interspecific incompatibility

Pre-zygotic interspecific incompatibility (II) involves an active inhibition mechanism between the pollen of one species and the pistil of another. As a barrier to fertilization, II effectively prevents hybridization and maintains species identity. Transgenic ablation of the mature transmitting tract (TT) in Nicotiana tabacum resulted in the loss of inhibition of pollen tube growth in Nicotiana obtusifolia (synonym Nicotiana trigonophylla) and Nicotiana repanda. The role of the TT in the II interaction between N.tabacum and N.obtusifolia was characterized by evaluating N.obtusifolia pollen tube growth in normal and TT-ablated N.tabacum styles at various post-pollination times and developmental stages. The II activity of the TT slowed and then arrested N.obtusifolia pollen tube growth, and was developmentally synchronized. We hypothesize that proteins produced by the mature TT and secreted into the extracellular matrix inhibit interspecific pollen tubes. When extracts from the mature TT of N.tabacum were injected into the TT-ablated style prior to pollination, the growth of incompatible pollen tubes of N.obtusifolia and N.repanda was inhibited. The classIII pistil-specific extensin-like protein (PELPIII) was consistently associated with specific inhibition of pollen tubes, and its requirement for II was confirmed through use of plants with antisense suppression of PELPIII. Inhibition of N.obtusifolia and N.repanda pollen tube growth required accumulation of PELPIII in the TT of N.tabacum, supporting PELPIII function in pre-zygotic II.

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.

Investigating mechanisms involved in the self-incompatibility response in Papaver rhoeas.

Sexual reproduction in flowering plants is controlled by recognition mechanisms involving the male gametophyte (the pollen) and the female sporophyte (the pistil). Self-incompatibility (SI) involves the recognition and rejection of self- or incompatible pollen by the pistil. In Papaver rhoeas, SI uses a Ca(2+)-based signalling cascade triggered by the S-protein, which is encoded by the stigmatic component of the S-locus. This results in the rapid inhibition of incompatible pollen tube growth. We have identified several targets of the SI signalling cascade, including protein kinases, the actin cytoskeleton and nuclear DNA. Here, we summarize progress made on currently funded projects in our laboratory investigating some of the components targeted by SI, comprising (i) the characterization of a pollen phosphoprotein (p26) that is rapidly phosphorylated upon an incompatible SI response; (ii) the identification and characterization of a pollen mitogen-activated protein kinase (p56), which exhibits enhanced activation during SI; (iii) characterizing components involved in the reorganization and depolymerization of the actin cytoskeleton during the SI response; and (iv) investigating whether the SI response involves a programmed cell death signalling cascade.

Mechanisms of self-incompatibility in flowering plants

Self-incompatibility is a widespread mechanism in flowering plants that prevents inbreeding and promotes outcrossing. The self-incompatibility response is genetically controlled by one or more multi-allelic loci, and relies on a series of complex cellular interactions between the self-incompatible pollen and pistil. Although self-incompatibility functions ultimately to prevent self-fertilization, flowering plants have evolved several unique mechanisms for rejecting the self-incompatible pollen. The self-incompatibility system in the Solanaceae makes use of a multi-allelic RNase in the pistil to block incompatible pollen tube growth. In contrast, the Papaveraceae system appears to have complex cellular responses such as calcium fluxes, actin rearrangements, and programmed cell death occurring in the incompatible pollen tube. Finally, the Brassicaceae system has a receptor kinase signalling pathway activated in the pistil leading to pollen rejection. This review highlights the recent advances made towards understanding the cellular mechanisms involved in these self-incompatibility systems and discusses the striking differences between these systems.

Selective inhibition of the growth of incompatible pollen tubes by S-protein in the Japanese pear

The S-allele-associated proteins (S-proteins) in the styles of the Japanese pear (Pyrus serotina Rehd. var. culta Rehd.) were purified by cation exchange chromatography. Their inhibitory action on the growth of incompatible pollen tubes (pollen tubes bearing the same S- allele as in the style from which the S-proteins were prepared) was characterized in vitro. Germination and tube growth of self-pollen (pollen from the same cultivar from which the S-proteins were prepared) decreased dose-dependently when the S-protein was added to the medium. Tube length was reduced to 10% that of compatible pollen tubes (pollen tubes bearing the S-allele different from that in the style from which the S-proteins were prepared) at 1.5 µg µl1. S-proteins from Shinsui (S 4 S 5 ) also inhibited growth of cross-incompatible Kosui (S 4 S 5 ) pollen tubes, but not of compatible Chojuro (S 2 S 3 ) pollen tubes. After inactivation of RNase of the S- protein, the inhibitory action of the S-protein disappeared. These results indicate that the S-protein acts directly to inhibit growth of incompatible pollen tubes in Japanese pear styles, and that the RNase activity of the protein is essential for the biological function. However, small amounts of proteins that co-migrated with the S-protein may also play some roles in the inhibition. This is the first report on the selective inhibitory action of S-proteins in Rosaceae.

Correlation between the Accumulation of Stylar Canal Exudate and the Diminution of Self-incompatibility in Aging Flowers of Lilium longiflorum.

Self-incompatibility in Lilium longiflorum cv. Hinomoto diminishes with aging of the flower. The relation between the diminished self-incompatibility of the aged flower and stylar canal exudate in Lilium longiflorum was studied.Carbohydrate content in stylar canal exudate increased with aging of the flower, indicat-ing that it accumulated with time. During the aging process, the molecular weight distribu-tion and composition of the arabinogalactans, which are the main components of the canal exudate, did not change. When the canal exudates collected from both young and old flow-ers were injected into stylar canals, the growth of incompatible selfed pollen tubes was sig-nificantly promoted. The promotive effect of stylar constituents on pollen tube growth did not differ between those from young and old flowers. The growth of incompatible pollen tubes in the aged pistils was significantly reduced by flushing the stylar canal with water which removed the canal exudate.These results suggest that the diminution of self-incompatibility in aged flowers is attributa-ble to the accumulation of the secreted canal exudate which plays a nutritional role for pollen tube growth.

Self‐incompatibility in Papaver rhoeas: there is no evidence for the involvement of stigmatic ribonuclease activity

Abstract. Ribonuclease assays have revealed, in contrast to the self‐incompatibility (SI) system of Nicotiana alata, there is no detectable ribonuclease activity that correlates with the presence of the functional stigmatic S‐gene product in Papaver rhoeas. Thus, we have shown that the inhibition of incompatible pollen tube growth in P. rhoeas is not associated with ribonuclease activity. Furthermore, the finding that pollen from P. rhoeas, unlike that from N. alata, is insensitive to purified bovine pancreatic ribonuclease A at very high concentrations, suggests that the involvement of ribonucleases in the inhibition reaction of the SI response in P. rhoeas is unlikely. In addition, the level of ribonuclease activity in mature stigmas of P. rhoeas is very much lower than that in N. alata and significantly, the level of ribonuclease activity did not rise in conjunction with the developmental expression of SI. Therefore, as a result of these studies, we believe that SI in P. rhoeas does not involve ribonuclease activity.

Action of the Style Product of the Self-Incompatibility Gene of Nicotiana alata (S-RNase) on in Vitro-Grown Pollen Tubes.

The products of the S-locus expressed in female tissues of Nicotiana alata are ribonucleases (S-RNases). The arrest of growth of incompatible pollen tubes in styles may result from entry of the S-RNase into the pollen tube and degradation of pollen tube RNA. We investigated the action of isolated S-RNases on pollen tubes grown in vitro and found that S-RNase is taken up by the pollen without substantial alteration. The S-RNases inhibit incorporation of exogenously added radioactive amino acids into protein by the germinated pollen. The S-RNases also inhibit in vitro translation of pollen tube RNA in a wheat germ cell-free extract. We found no evidence for a specific mRNA substrate for the S-RNases, which implies that if RNase activity is involved in the control of self-incompatibility, allelic specificity is more likely to depend on the selective uptake of S-RNases into pollen tubes or their selective activation or inactivation by pollen factors, rather than cleavage of a specific substrate. Heat treating S2-RNase largely destroys its RNase activity but increases its inhibitory effect on in vitro pollen tube growth. This effect is not due to an increased uptake of S2-RNase by the pollen but is associated with a greatly enhanced accumulation of S2-RNase on the outer surface of the pollen grains.

Self‐Incompatibility in Papaver rhoeas L.: inhibition of incompatible pollen tube growth is dependent on pollen gene expression

summaryIn order to gain an insight into the cellular activities which lead to the inhibition of pollen tube growth following a self‐incompatible response, we have been studying the effects of various metabolic inhibitors on pollen–stigmatic extract interactions in vitro. The results indicate that both transcription and glycosylation are required for the full inhibition of pollen tube growth during an incompatible response in Papaver rhoeas. The ability of actinomycin D to partially alleviate an incompatible reaction suggests that during the response, pollen gene expression is induced; we have found that this is indeed the case and have identified novel proteins produced in the pollen which are associated with the incompatibility response. These findings give a clear indication that de novo transcription of pollen genes which are specific to this response, play an important role in the inhibition of pollen tube growth in this species. This provides a significant step towards the elucidation of the mechanism whereby pollen tube growth is arrested following an incompatible reaction in this species.

Sequence variability and developmental expression of S-alleles in self-incompatible and pseudo-self-compatible petunia.

We investigated the structure and expression of three S-alleles of Petunia hybrida in self-incompatible varieties and in a pseudo-self-compatible line in which the self-incompatibility response is defective. Comparison of derived amino acid sequences from different gametophytic S-alleles revealed a pattern of sequence conservation and variability that was highly nonrandom. In self-incompatible varieties, petunia S-locus mRNA accumulates preferentially in styles during the transition from bud self-compatibility to self-incompatibility. S-Allele sequences homologous to the cloned S1 allele were present in a pseudo-self-compatible variety, and were expressed at levels indistinguishable from those observed in a self-incompatible line homozygous for the S1 allele. Taken together, our data indicate that (1) limited sequence differences may confer allelic specificity, (2) S-locus mRNAs accumulate in a precise organ-specific pattern during floral development, and (3) the ability to inhibit the growth of incompatible pollen tubes appears to require a threshold accumulation of the stylar gene product, along with the participation of as yet undefined pollen gene products.

Incompatible pollen tube growth and protein composition in styles of Japanese pear following high temperature treatments

Immersing the excised-prepollinated styles of self-incompatible Japanese pear, Pyrus serotina Rehd., into distilled water at 45°C for 1.5–2.5 min promoted the growth of self-pollen tubes; both the number of styles penetrating the pollen tubes through styles to cut ends and of pollen tubes protruding from cut ends were increased. The subsequent growth of the protruded pollen tubes on agar media was also enhanced by the treatments. Treatments at 40°C did not promote the growth of self-pollen tubes and those at 50°C led the styles to necrosis. In the stylar proteins analyzed by isoelectricfocusing polyacrylamide gel electrophoresis, several changes in banding pattern were found following the heat treatment; one of the bands was a glycoprotein that increases in quantity in developing styles as self-incompatibility of the style becomes stronger (Hiratsuka et al., 1986). This protein band was stained more weakly by Coomassie Brilliant Blue-G250 dye. Antigenic substances in the styles examined by immunodiffusion seemed not to be denatured by high temperature treatments.

Analysis of Self-incompatibility Reaction in Easter Lily by Using Heat Treatments

Abstract Self-incompatibility of Easter lily (Lilium longiflorum Thunb.) was overcome by heating the styles. Immersing detached prepollinated styles in distilled water at 45C for 5 min promoted the growth of incompatible pollen tubes. When heat-treated styles were kept at 25C for various periods before pollination, the effect of overcoming selfincompatibility diminished as the period between the heat treatment and pollination was increased. Heat treatment of styles within 2 hr after incompatible pollination promoted the growth of pollen tubes to a distance comparable to compatible ones. When the styles were heated during the period from 3 to 8 hr after incompatible pollination, the growth of pollen tubes was retarded to some extent as compared to those treated within 2 hr after pollination. Treatment at 12 or more hours after pollination no longer promoted the growth of incompatible pollen tubes. These results suggest that overcoming self-incompatibility by heating is temporary and the incompatibility machinery is already induced at ≈3 hr after pollination.

テッポウユリの雌ずい物質が花柱内での花粉管伸長に及ぼす影響と受粉による花柱内物質の変動

When stigmatic sap or stylar sap collected by centrifugation was placed on the stylar canals of bisected pistils of Lilium longiflorum, or when pollen grains were pre-soaked in the stigmatic sap, growth of pollen tubes in the style was accelerated even inn self-combination.When stylar sap collected from cross-pollinated styles was placed on the stylar canals of the bisected pistils, the growth of incompatible pollen tubes was significantly promoted more than by the placement of the sap from non- or self-pollinated styles.Changes in constituents of the stylar sap after pollination were also examined. Protein content in the sap decreased in the upper 4/5 of the style in 24 hrs after pollination. Stylar sap eluted through Sephadex G-150 column showed two peaks of carbohydrates, i.e., a broad band of high molecular weight and a narrow band of low molecular weight. After cross-pollination, both peaks were lowered, while after self-pollination only the peak of low molecular weight was lowered.These results suggest that the movement of stylar sap from the stylar tissue to the stylar canal is different between self- and cross-combinations, and that the inhibitory substance for the pollen tubes is absent even in self-combination, and that certain proteoglycans are related to self-incompatibility.

テッポウユリの異品種の花柱および前受粉した雌ずいの花柱を組合せて接合した花柱内における花粉管伸長

The self-incompatibility in Lilium longiflorum was investigated using the differently grafted pistils. Pistils of Lilium longiflorum cv. Georgia and Hinomoto were cut transversely into two parts at 15 or 25mm from stigma tips. They were then grafted in different combinations with silicone rubber tube (2.5mm in inner diameter). Pollen tube growth in the grafted pistils was somewhat reduced as compared with that in the intact style, but the incompatibility responses of the pollen tubes could be studied in the grafted styles.In the pistils grafted with different cultivar, the appearance of self-incompatible responses was observed on the style from the same cultivar. The ovary end portion of the grafted styles seemed to be more influential.In the pistils grafted with cross-pre-pollinated pistil segment, the incompatible pollen tube growth was faster than in the pistils grafted with non- or self-pre-pollinated pistil segments. The necessary condition for the rapid growth of pollen tube was generated in the styles after cross-pollination, with a qrobable velocity of 0.5cm/h from the stigma to the ovary.In self-pre-pollinated pistils, the necessary condition for the rapid growth of pollen tube seemed not to be generated in the style and further any inhibitor for the pollen tube growth was not produced.

Studies on the self-incompatibility of Petunia hybrida in excised-style culture. Differences in self-incompatibility reaction among four clones

In Petunia clones with different S-alleles, self- or cross-pollinated excised-styles of 5, 10, 15, 20 or 25 mm were incubated on a standard agar medium for 24, 48 or 72 h. The length and number of protruding incompatible pollen tubes were compared with those of compatible ones. Throughout the experimental period, the length and number of incompatible pollen tubes of pollen from the S1S1-clone were always less than those of compatible ones. In pollen from S2S2-, S3S3- and S2S3-clones the incompatible pollen tube growth was barely arrested in 5 and 10 mm excised-styles during the first 24 h of incubation. However, inhibition of incompatible pollen tube growth was strengthened with the increase of both excised-style length and incubation period: this was clearly evident in 15 mm or longer excised-styles incubated for 48 h. Ratios of incompatible to compatible pollen tube length in excised-styles incubated for 72 h, were for S3S3 pollen tubes = 0.28, S1S1=0.48, S2S3 = 0.50, and S2S2 = 0.60, and ratios on tube numbers were S3S3 = 0.01, S1S1 = 0.1, S2S2 = 0.21, and S2S3 = 0.21. These results were in agreement with those of in vivo self-pollination. The incompatibility reaction seemed strongest in S3S3-, weaker in S1S1 - and weakest in S2S2- and S2S{3}-clones, and therefore the intensity of S-allele expression would be S3> S1> S2.