Adhesion and guidance in compatible pollination

The mechanisms of compatible pollination are less studied than those of incompatible pollination and yet most of the angiosperms show self-compatibility. From the release of pollen from anthers to the penetration of the micropyle by the pollen tube tip, there are numerous steps where the interaction between pollen and the pistil can be regulated. Recent studies have documented some diverse ways in which pollen tubes carrying sperm cells are guided to the ovules through the pistil extracellular matrices of the transmitting tract. What is still missing is an understanding of pollen tube cell biology in vivo. A recent finding supports the role of the synergids in the crucial guidance cue for the pollen tube tip at the micropyle, but experimental evidence for other 'guidepost' cells in the pistil is still lacking. The fact that the pollen tube must first travel through the matrices of the stigma and style before it can respond to the cue from the ovule makes it likely that there is a hierarchy of signalling events in pollen-pistil interactions starting at the stigma and ending at the micropyle. On the pistil side, several model systems have been used in the discovery of molecules implicated in either physical or chemical guidance. In lily, which has a hollow style, adhesion molecules (pectin and SCA) are implicated in guidance. SCA alone is also capable of inducing pollen chemotropism in an in vitro assay, suggesting that this peptide plays a dual role in lily pollination: chemotactic in the stigma and haptotactic (adhesion mediated) in the style.     

Pollen-pistil interaction in kiwifruit (Actinidia deliciosa; Actinidiaceae)

The pollen-pistil interaction has been examined in kiwifruit (Actinidia deliciosa). In this species a large number of seeds are produced in each fruit and a great many pollen grains germinate and grow to reach the ovules. This growth is assisted by an abundant secretion that is present all along the pistilar tract. At anthesis, the stigma is covered by a secretion where the pollen grains germinate and grow. The stylar transmitting tissue is initially rich in starch reserves, but the starch gradually disappears and, concomitantly, an abundant secretion that stains for carbohydrates appears in all of the intercellular spaces. Pollen tube growth relies on this secretion since it is depleted after pollen tube passage, while in unpollinated flowers it remains unaltered throughout the flower life-span. In the ovary a similar situation occurs. The placental surface, where the pollen tubes grow before reaching the ovules, is covered by a number of obturators. At anthesis, these obturators are rich in starch reserves and have an abundant secretion on their outer surface. As time passes, starch disappears while the secretion increases. It is in this secretion that the pollen tubes grow on their way toward the ovules. These observations are discussed in terms of the support given by the pistil to pollen tube growth to achieve the highly successful reproductive performance of this species.     

Pollen tubes enter neighbouring ovules by way of receptacle tissue,resulting in increased fruit-set in Sagittaria potamogetifolia Merr

Using fluorescence microscopy, deposition of pollen on stigmas and pollen tube growth in the gynoecium of Sagittaria potamogetifolia Merr., a monoecious species with an apocarpous gynoecium, were observed. The maximum rate of pollination averaged 83.9 +/- 4.7 %, and the number of pollen grains per stigma ranged from zero to 30. Pollen tubes grew through one stigma to the base of the ovary at almost the same speed, but generally only one of the pollen tubes then turned towards the ovule and finally entered the nucellus through the micropyle. The other pollen tubes grew through the ovary base and the receptacle tissue into ovules of adjacent carpels whose stigmas were not pollinated or which had been pollinated later. This phenomenon is termed pollen tube 'reallocation' by the authors. To verify the direct effect of the phenomenon on fruit set, artificial pollination experiments were conducted in which two or more pollen grains were placed onto only one stigma in each gynoecium; frequently more than one fruitlet was obtained from each flower treated. The reallocation of pollen tubes among pistils in the gynoecium could effect fertilization of ovules of unpollinated pistils and lead to an increase in sexual reproduction efficiency. It would, to some extent, also increase pollen tube competition among pistils of the whole gynoecium.     

Arabinogalactan-Proteins of the Female Sexual Tissue of Nicotiana alata: I. Changes during Flower Development and Pollination

Arabinogalactan-proteins (AGPs), isolated from the pistils of Nicotiana alata, an ornamental tobacco, are developmentally regulated. Both the total amount and concentration of AGP in the stigma increase during flower development, reaching 10 micrograms AGP/stigma at maturity. In contrast, AGP concentration in the style remains constant throughout the maturation period reaching 12 micrograms AGP/style at maturity. The classes of AGP present in the stigma and style during flower development, separated according to their charge by crossed-electrophoresis, are different and change during development. Pollination of flowers of N. alata with compatible or incompatible pollen results in a significant and reproducible increase in the amount of AGPs in the stigma, but not the style, compared with control unpollinated pistils. Pollination with ethanol vapor inactivated pollen also results in an increase in the amount of AGP in the stigma, but this is less than half that observed following pollination with viable pollen. There are no significant differences in the classes of AGP, based on crossed-electrophoresis, present in the pistil following pollination.     

Changes in proteins and peroxidases induced by compatible pollination in the ovary of Nicotiana tabacum L. ahead of the advancing pollen tubes

Proteins and peroxidases produced by the ovules and placenta of tobacco (Nicotiana tabacum L.) in response to compatible pollination were analyzed by two-dimensional polyacrylamide gel electrophoresis and by enzyme staining in flat-bed native isoelectric focusing gels. For two-dimensional gels, ovaries were sampled at 36 h after pollination, at which time pollen tubes have penetrated much of the length of the style but have not yet entered the ovary. At least 11 major proteins from pollinated ovaries had no detectable counterparts in unpollinated ovaries. These showed a range of molecular mass and pI. For peroxidase isozyme assays, ovaries were sampled at 0, 12, 24, 36 and 48 h after pollination. At 45–50 h, pollen tubes were beginning to enter the top of the ovary but could still be separated from the ovules and placenta during sampling. Ovules and placentae from unpollinated pistils showed only one form of peroxidase, whereas those from pollinated pistils showed additional isozymes at pH 5.4 and pH 10.0. Both new isozymes increased in staining intensity over the first 36 h after pollination. At 48 h, however, the acidic peroxidase had continued to increase, while the basic component had declined so as to be barely detectable. The observations are discussed in relation to accumulating evidence that some form of pollination-induced signal reaches the ovary ahead of the advancing pollen tubes. The nature of this signal and possible involvement of peroxidases are also briefly discussed.     

Pollen tube growth and guidance: roles of small, secreted proteins

BACKGROUND: Pollination is a crucial step in angiosperm (flowering plant) reproduction. Highly orchestrated pollen-pistil interactions and signalling events enable plant species to avoid inbreeding and outcrossing as a species-specific barrier. In compatible pollination, pollen tubes carrying two sperm cells grow through the pistil transmitting tract and are precisely guided to the ovules, discharging the sperm cells to the embryo sac for fertilization. SCOPE: In Lilium longiflorum pollination, growing pollen tubes utilize two critical mechanisms, adhesion and chemotropism, for directional growth to the ovules. Among several molecular factors discovered in the past decade, two small, secreted cysteine-rich proteins have been shown to play major roles in pollen tube adhesion and reorientation bioassays: stigma/style cysteine-rich adhesin (SCA, approx. 9·3 kDa) and chemocyanin (approx. 9·8 kDa). SCA, a lipid transfer protein (LTP) secreted from the stylar transmitting tract epidermis, functions in lily pollen tube tip growth as well as in forming the adhesive pectin matrix at the growing pollen tube wall back from the tip. Lily chemocyanin is a plantacyanin family member and acts as a directional cue for reorienting pollen tubes. Recent consecutive studies revealed that Arabidopsis thaliana homologues for SCA and chemocyanin play pivotal roles in tip polarity and directionality of pollen tube growth, respectively. This review outlines the biological roles of various secreted proteins in angiosperm pollination, focusing on plant LTPs and chemocyanin.     

Low genetic diversity and functional constraint of miRNA genes participating pollen–pistil interaction in rice

Key message

In this study, we sequenced and analyzed the expression and evolution of rice miRNA genesparticipating pollen-pistil interaction that is crucial to rice yield.

Abstract

Pollen–pistil interaction is an essential reproductive process for all flowering plants. While microRNAs (miRNAs) are important noncoding small RNAs that regulate mRNA levels in eukaryotic cells, there is little knowledge about which miRNAs involved in the early stages of pollen–pistil interaction in rice and how they evolve under this conserved process. In this study, we sequenced the small RNAs in rice from unpollinated pistil (R0), pistil from 5 min and 15 min after pollination, respectively, to identify known and novel miRNAs that are involved in this process. By comparing the corresponding mRNA-seq dataset, we identified a group of miRNAs with strong negative expression pattern with their target genes. Further investigation of all miRNA loci (MIRNAs) across 1083 public rice accessions revealed significantly reduced genetic diversity in MIRNAs with strong negative expression of their targets when comparing to those with little or no impact on targets during pollen–pistil interaction. Annotation of targets suggested that those MIRNAs with strong impact on targets were pronounced in cell wall related processes such as xylan metabolism. Additionally, plant conserved miRNAs, such as those with functions in gibberellic acid, auxin and nitrate signaling, were also with strong negative expression of their targets. Overall, our analyses identified key miRNAs participating pollen–pistil interaction and their evolutionary patterns in rice, which can facilitate the understanding of molecular mechanisms associated with seed setting.

     

The reproductive biology of Cybistax antisyphilitica (Bignoniaceae), a characteristic tree of the South American savannah-like “Cerrado” vegetation

Bignoniaceous woody species are very important ecological components of neotropical forests, but the reproductive biology of many species, such as Cybistax antisyphilitica, remains virtually unknown. Most species of Bignoniaceae are characteristically self-sterile, despite typically exhibiting normal pollen tube growth throughout the style, combined with slow rates of ovule penetration, fertilisation and endosperm initiation in selfed pistils. Uniform abortion occurs within a few days of anthesis, indicating the occurrence of late-acting self-incompatibility (LSI). However, breeding system studies have been performed in fewer than 7% of species, and other types of breeding systems (e.g., self-compatibility and apomixis) have been reported in this family. In the present study, the reproductive biology of C. antisyphilitica was investigated by field observation of flower visitors and floral events. Moreover, reproductive biology of this species was examined through experimental pollinations, analyses of pollen tube growth and ovule penetration using fluorescence microscopy, verification of pistil longevity, and a histological analysis of unpollinated vs. self-pollinated pistils. Finally, morphological aspects, quantities and germination were investigated in seeds that resulted from different pollination treatments. Natural pollination was effected by large- and medium-sized bees, and their visiting behaviour favours a high proportion of geitonogamy and no pollen limitation. Self-pollinated flowers produced no fruits, and all of the characteristic post-pollination events cited above were verified, witnessing the occurrence of LSI with post-zygotic rejection of selfed pistils in C. antisyphilitica. Although some indications of extended pistil longevity were found in selfed pistils, this feature seemed to be affected by unidentified environmental factors. The seeds were always monoembryonic and with high viability. A larger variation in the number of viable seeds was found in fruits derived from natural pollination. A low fruit set was observed after both natural and cross-pollination, and most crossed fruits underwent abortion at several points during the juvenile phase, even when protected against herbivory. The formation of surplus flowers/juvenile fruits and the apparently wasteful selfing mechanism control implied in LSI are discussed in the context of the perennial life style of tropical woody plant species.     

Delayed fertilization and pollen-tube growth in pistils of Fagus japonica (Fagaceae)

In contrast to most angiosperms, in which fertilization occurs 1 or 2 days after pollination, in some plant orders, including the Fagales, fertilization is delayed from 4 days to more than 1 year, raising questions regarding why fertilization is delayed and where and how pollen tubes remain in the pistil during the delay. To answer these questions, we investigated pollen-tube growth in pistils of Fagus japonica (Fagaceae), which are tricarpellate and have six ovules, using light, fluorescence, and scanning electron microscopy. The ovules were immature at the time of pollination and required 5 weeks to become fully developed. During this 5 weeks, pollen tubes grew from the stigma to the embryo sac in association with the development of ovules and intermittently in three steps with two growth-cessation sites, i.e., on the funicle and near the micropyle. The number of pollen tubes was gradually reduced from many to one at the two growth-cessation sites, and fertilization occurred in one ovule that apparently developed earlier than the others in the pistil. Thus, delayed fertilization plays an important role in gametophyte competition and selection leading to nonrandom fertilization. Intermittent pollen-tube growth is also likely widespread in angiosperms because it is known in other Fagales and an unrelated order Garryales.     

Sulfinylated azadecalins act as functional mimics of a pollen germination stimulant in Arabidopsis pistils

Polarized cell elongation is triggered by small molecule cues during development of diverse organisms. During plant reproduction, pollen interactions with the stigma result in the polar outgrowth of a pollen tube, which delivers sperm cells to the female gametophyte to effect double fertilization. In many plants, pistils stimulate pollen germination. However, in Arabidopsis, the effect of pistils on pollen germination and the pistil factors that stimulate pollen germination remain poorly characterized. Here, we demonstrate that stigma, style, and ovules in Arabidopsis pistils stimulate pollen germination. We isolated an Arabidopsis pistil extract fraction that stimulates Arabidopsis pollen germination, and employed ultra‐high resolution electrospray ionization (ESI), Fourier‐transform ion cyclotron resonance (FT‐ICR) and MS/MS techniques to accurately determine the mass (202.126 Da) of a compound that is specifically present in this pistil extract fraction. Using the molecular formula (C₁₀H₁₉NOS) and tandem mass spectral fragmentation patterns of the m/z (mass to charge ratio) 202.126 ion, we postulated chemical structures, devised protocols, synthesized N‐methanesulfinyl 1‐ and 2‐azadecalins that are close structural mimics of the m/z 202.126 ion, and showed that they are sufficient to stimulate Arabidopsis pollen germination in vitro (30 μm stimulated approximately 50% germination) and elicit accession‐specific response. Although N‐methanesulfinyl 2‐azadecalin stimulated pollen germination in three species of Lineage I of Brassicaceae, it did not induce a germination response in Sisymbrium irio (Lineage II of Brassicaceae) and tobacco, indicating that activity of the compound is not random. Our results show that Arabidopsis pistils promote germination by producing azadecalin‐like molecules to ensure rapid fertilization by the appropriate pollen.     

Pollen-pistil interactions and early fruiting in parthenocarpic citrus

Background and Aims

An intense pollen–pistil interaction precedes fertilization. This interaction is of particular relevance in agronomically important species where seeds or fruits are the edible part. Over time some agronomically species have been selected for the ability to produce fruit without seeds. While this phenomenon is critical for commercial production in some species, very little is known about the events behind the production of seedless fruit. In this work, the relationship between pollen–pistil interaction and the onset of fruiting was investigated in citrus mandarin.

Methods

Pistils were sequentially examined in hand-pollinated flowers paying attention to pollen-tube behaviour, and to cytochemical changes along the pollen-tube pathway. To evaluate which of these changes were induced by pollination/fertilization and which were developmentally regulated, pollinated and unpollinated pistils were compared. Also the onset of fruiting was timed and changes in the ovary examined.

Key Results

Conspicuous changes occurred in the pistil along the pollen-tube pathway, which took place in a basipetal way encompassing the timing of pollen-tube growth. However, these changes appear to be developmentally regulated as they happened in the same way and at the same time in unpollinated flowers. Moreover, the onset of fruiting occurred prior to fertilization and the very same changes could be observed in unpollinated flowers.

Conclusions

Pollen–pistil interaction in citrus showed similarities with unrelated species and families belonging to other taxa. The uncoupling of the reproductive and fruiting processes accounts for the parthenocarpic ability of unpollinated flowers to produce fruit in citrus. However, the maintenance of a functional reproductive process reflects the potential to produce seeded fruits, providing a basis for the understanding of the production of seeded or unseeded fruits and further understanding of the process of parthenocarpy in other species.     

The Nutritional Role of Pistil Exudate in Pollen Tube Wall Formation in Lilium longiflorum: II. Production and Utilization of Exudate from Stigma and Stylar Canal

Detached pistils of Lilium longiflorum were labeled with d-glucose-U-¹⁴C 24 hours after anthesis and then sampled for the next 6 days to determine the appearance of label into exudate from the stylar canal and the stigmatic surface of the pistil. Results were obtained with unpollinated cv. Ace pistils and pollinated cv. Ace pistils, selfed or crossed (cv. Croft pollen). Limited data were also obtained on cv. Croft pistils, selfed or crossed (cv. Ace pollen).     

Influences of Cross Pollination on Pollen Tube Growth and Fruit Set in Zuili Plums （Prunus salicina）

Zuili plum （Prunus salicina L.） trees usually set fruit poorly, although they produce high quality fruit. To elucidate the causes of the poor fruit set, pollen tube growth into pistils and fruit set percentage were investigated after cross-, self- and open-pollination. Ovule development in Zuili pistils was also investigated. Pollen tube penetration into the ovules via the obturator and micropyle was best when Zuili pistils were pollinated by cv. Black Amber （P. domestica） pollen grains, although cross-pollinations with Hongxinli and Miili （P. salicina） pollen were more effective than self- and open-pollination. The fruit set percentage was also highest in pistils pollinated with Black Amber pollen grains. Morphological observation of Zuili pistils revealed that the trees produce ＂double pistils＂, developing two ovaries from a basal pistil, at a rate as high as 28%. In such abnormal pistils, most ovules were lacking an embryo sac or were entirely degenerated. The percentage of normally developed ovules was 24.3% and 8.9% in normal and double pistils, respectively. From these results, we conclude that the main causes of poor fruit set of Zuili plums are a lack of effective cross-pollination and the production of high percentages of double pistils in which normally developed ovules are scarcely formed.     

Pollen-tube growth pattern and chalazogamy in<Emphasis Type="Italic"> Casuarina equisetifolia</Emphasis> (Casuarinaceae)

For a better understanding of pollen-tube guidance in relation to pollen-pistil interaction, we investigated the mode of pollen-tube growth in pistils of Casuarina equisetifolia, a monoecious, wind-pollinated species that undergoes chalazogamous fertilization. The pistil is bicarpellate, but only one of the two carpels develops with two ovules. One of these ovules develops more than four embryo sacs. Pistils usually require more than 1 month to reach maturity after pollen grains have been deposited on the stigmas. During that period, pollen-tube growth proceeds discontinuously in five distinct steps that lead up to fertilization: (1) from the stigma to the upper region of the style, (2) from the upper region of the style to a septum in the ovary, (3) from the septum to the surface of the funiculus, (4) from the funiculus to chalaza in the ovule, and (5) from the chalaza to an egg apparatus. Probably because of competitive interaction between male and female gametophytes (or ovules), one pollen tube is selected from among many during the first step (just before the second step), one ovule from the two during the second and third steps, and one embryo sac from more than four during the fourth and fifth steps. On the basis of our results, erroneous drawings and explanations reported in earlier publications on chalazogamy in Casuarinaceae should be brought into question.     

COORDINATED TIMETABLES FOR MEGAGAMETOPHYTE DEVELOPMENT AND POLLEN TUBE GROWTH IN RHODODENDRON NUTTALLII FROM ANTHESIS TO EARLY POSTFERTILIZATION

Rhododendron nuttallii T. W. Booth (Ericaceae) was used to derive concurrent timetables for megagametophyte, pollen tube and early postfertilization development from anthesis through 3 wk after pollination, based on timed collections of self-pollinated pistils. Stages of development were determined for over 33,500 cleared ovules, including, for selected collection dates, stages on different portions of the placenta. Pollen tube information was obtained by fluorescence microscopy of pistil squashes stained with aniline blue. Because of the very large number of ovules observed, it was possible to recognize a much more closely graded series of stages in megagametophyte development than is usually the case. While a range of stages occurred on all days, development progressed steadily from a majority of functional spores and 2-nucleate gametophytes on the day of anthesis to mostly a late zygote-primary endosperm stage at 18 days, and some 2-celled endosperm stages at 21 days, after pollination. At all times the most advanced stages, including first pollen tube entries, occurred on the outer surface of the lower half of the placenta, and the youngest on the inner surface of the uppermost portion. Fertilizable ovules were not found in any frequency until 8 days after pollination (then in only about 34% of the ovules); a few fertilized ones were seen after 10 days but constituted less than 5% until 12 days after pollination, thereafter increasing to about 60%. Fertilization occurred in any one of three morphologically recognizable stages distinguished by position and state of fusion of polar nuclei. Pollen germinated on the stigma 1–2 hr after pollination, and pollen tubes grew at a rate of about 1–1.25 cm/day, reaching the top of the ovary in 8–9 days with the first ovule entries seen after 10 days. There was a close correlation between megagametophyte development and pollen tube growth, with large numbers of functionally mature ovules not being found until pollen tubes had reached the ovary. While nuclei within ovules could not be distinguished in the squashes, three gametophyte stages that could be recognized—unelongated, elongated either without or with a pollen tube—were tallied for almost 29,000 ovules. The progression in these general stages corresponded well with that documented in more detail from cleared ovules. Unpollinated pistils showed a similar progression of gametophyte stages until the time fertilization would start to occur, after which there was continued accumulation of functionally mature ovules. A variety of abnormally developed and/or collapsing(ed) ovules or gametophytes were seen; collectively, they averaged over 8.6% of all ovules.     

Isolation of Tissue-Specific cDNAs from Tomato Pistils

We have used a differential plaque hybridization screening procedure to isolate cDNA clones for genes that show elevated or exclusive expression in tomato pistils. Clones that showed maximal expression in immature pistils (premeiotic to early meiosis) and mature pistils (at anthesis) were isolated. Of nine clones that were characterized, four were found also to express at some stage of anther development. In situ hybridization experiments showed that expression of the genes we have identified is very tightly regulated both spatially and temporally within the pistil. One gene was identified that is expressed in the pistil only in the transmitting tissue of the style. A second gene was found to express exclusively in two to three cell layers of the ovules for a period of less than eight days.     

Effects of self-, chase and mixed self/cross-pollinations on pistil longevity and fruit set in Ceiba species (Bombacaceae) with late-acting self-incompatibility

BACKGROUND AND AIMS: Late-acting self-incompatibility (LSI), in which selfed flowers fail to form fruits despite apparently successful growth of the pollen tubes to the ovules, is a contentious and still poorly understood phenomenon. Some studies have indicated pollen tube-pistil interactions, and major gene control. Others favour an early acting inbreeding depression explanation. METHODS: Experimental pollinations, including selfs (in a subsample of which the style was cut before pollen tubes reached the ovary), chase self/cross-pollinations, crosses, and mixed self/cross-pollinations were used to study floral/pistil longevity and effect on fruit set and seed yield in two Ceiba species known to have LSI. RESULTS: Self-pollinations, including those with a cut style, had extended floral longevity compared with unpollinated flowers. Chase pollinations in which cross-pollen was applied up to 3 h after selfing set fruits, but with reduced seed set compared with crosses. Those with cross-pollen applied at 4 and 8 h after self-pollination all failed to set fruits. Flowers subjected to 1 : 1 and 2 : 1 self/cross-pollinations all produced fruits but again with a significantly lower seed set compared with crosses. CONCLUSIONS: Extended floral longevity initiated with self-pollen tubes growing in the style indicates some kind of pollen tube-pistil interaction. Fruit set only in chase pollinations up to 3 h implies that self-pollen tubes either grow more slowly in the style or penetrate ovules more slowly on arrival at the ovary compared with cross-tubes. This agrees with previous observations indicating that the incidence of penetrated ovules is initially lower in selfed compared with crossed pistils. However, the low seed yield from mixed pollinations indicates that self- and cross-pollen tubes arrive at the ovary and penetrate ovules more or less simultaneously. Possible explanations for these discordant results are discussed.