Arabidopsis pollen prolyl-hydroxylases P4H4/6 are relevant for correct hydroxylation and secretion of LRX11 in pollen tubes.

Major constituents of the plant cell walls are structural proteins that belong to the hydroxyproline-rich glycoprotein (HRGP) family. Leucine-rich repeat extensin (LRX) proteins contain a leucine-rich domain and a C-terminal domain with repetitive Ser-Pro3-5 motifs that are potentially to be O-glycosylated. It has been demonstrated that pollen-specific LRX8-LRX11 from Arabidopsis thaliana are necessary to maintain the integrity of the pollen tube cell wall during polarized growth. In HRGPs, including classical extensins (EXTs), and probably in LRXs, proline residues are converted to hydroxyproline by prolyl-4-hydroxylases (P4Hs), thus defining novel O-glycosylation sites. In this context, we aimed to determine whether hydroxylation and subsequent O-glycosylation of Arabidopsis pollen LRXs are necessary for their proper function and cell wall localization in pollen tubes. We hypothesized that pollen-expressed P4H4 and P4H6 catalyze the hydroxylation of the proline units present in Ser-Pro3-5 motifs of LRX8-LRX11. Here, we show that the p4h4-1 p4h6-1 double mutant exhibits a reduction in pollen germination rates and a slight reduction in pollen tube length. Pollen germination is also inhibited by P4H inhibitors, suggesting that prolyl hydroxylation is required for pollen tube development. Plants expressing pLRX11::LRX11-GFP in the p4h4-1 p4h6-1 background show partial re-localization of LRX11-green fluorescent protein (GFP) from the pollen tube tip apoplast to the cytoplasm. Finally, immunoprecipitation-tandem mass spectrometry analysis revealed a decrease in oxidized prolines (hydroxyprolines) in LRX11-GFP in the p4h4-1 p4h6-1 background compared with lrx11 plants expressing pLRX11::LRX11-GFP. Taken together, these results suggest that P4H4 and P4H6 are required for pollen germination and for proper hydroxylation of LRX11 necessary for its localization in the cell wall of pollen tubes.

Light and electron microscopies reveal unknown details of the pollen grain structure and physiology from Brazilian Cerrado species.

Pollen grains have a relatively simple structure and microscopic size, with two or three cells surrounded by the protective sporoderm at maturity. The viability and efficiency of pollen transport from anther to stigma depends on pollen physiological properties, especially the relative water content of the vegetative cell. Pollen transport is a crucial fate for most angiosperms that depends on biotic pollinators and studies focusing on understanding the morpho-physiological properties of pollen grains are still scarce, especially to tropical open physiognomies as the Brazilian Cerrado. Therefore, we investigate some structural and physiological aspects of pollen grains from six native species naturally growing in one Cerrado area: Campomanesia pubescens (Myrtaceae), Caryocar brasiliense (Caryocaraceae), Erythroxylum campestre (Erythroxylaceae), Lippia lupulina (Verbenaceae), Pyrostegia venusta (Bignoniaceae), and Xylopia aromatica (Annonaceae). We selected dehiscent anthers and mature pollen grains to analyze (1) the anther wall and pollen microstructure, (2) the pollen water status at the time of anther dehiscence, and (3) the pollen chemical compounds. In all analyzed species, the anther and pollen developed in a successfully way, and except for Caryocar brasiliense, all species were able to emit pollen tubes in the germination tests. As expected for a dry and open environment, most species dispersed their pollen grains in a partially dehydrated form, as indicated by our harmomegathy experiment. As indicated by our study, the pollen ability in preventing dissection, maintaining its viability in a dry and hot environment during its transport from anther to stigma, may be related to the sporoderm apertures and to the reserve compounds, mainly carbohydrates in the form of hydrolysable starch grains.

Pollen morphology and viability of Tillandsia (Bromeliaceae) species by light microscopy and scanning electron microscopy.

Tillandsia is the bromeliad genus containing the largest number of species, with wide geographic dispersion and an important ecological role in the ecosystems. Investigations of pollen morphology are important to support taxonomic and conservation studies as well as to choose promising species for cross-pollination to obtain hybrids. The objective of this study was to evaluate the morphology, production, and viability of pollen grains of 24 Tillandsia species that naturally occur in the state of Bahia, Brazil. Pollen grains were acetolized and analyzed by scanning electron microscopy. The pollen were germinated in vitro in two culture media (BM and BKM) and collected at three floral development stages (pre-anthesis, anthesis and post-anthesis). Pollen viability also was analyzed by staining with Alexander's solution and acetocarmine in three floral development stages. Variations in the ornamentation and size of the pollen were observed among the species studied, with the majority having medium size, while T. polystachia and T. juncea had large grains and the subgenera Diaphoranthema and Phytarrhiza had small grains. The pollen of the majority of species had bilateral symmetry, with a single irregularly shaped colpus, semitectate exine and reticulated-heterobrochate surface. The pollen presented high germination percentage, tube length and viability according to the histochemistry, except for T. recurvata, T. usneoides and T. loliacea. The BKM medium and collection of pollen during anthesis produced the best results for the Tillandsia species. The results can support studies of the taxonomy and conservation of these species, which have great environmental importance and high value for ornamental purposes.

Cross pollination of different peony cultivars with 'Feng dan' / Polinização cruzada de diferentes cultivares de peônia com 'Fengdan'

Paeonia ostii ‘Feng dan’ is recommended as a new promising oil crop because of the nutrient elements in its seed; however, the development of this new oil crop is now limited due to its low seed setting rate. This study identified ways to improve the cross pollination of Feng dan as the maternal parent, which has been proved to be a suitable oil crop, so as to improve the seed setting rate of ‘Feng dan’, and to predict the functional relationship between pollen vigor and seed setting rate. improving the efficiency of cross pollination. Three major steps were conducted, first, to screen the appropriate method of measuring pollen vigor of the paternal parent, which could have a strong relationship with seed setting rate. Second, to obtain the corresponding seed setting rate by cross pollination. Third, the functional relationship between two indexes was founded which could be used in cross pollination practice. Results indicated that the best paternal parents of ‘Feng dan’ were ‘Feng dan’ cultivars from other different cultivation populations; The most suitable medium concentration for pollen germination was 100~150 g/L sucrose, 0.06~0.08 g/L boric acid, and 50~200 g/L PEG-6000. Both the seed setting rate and the number of seed grains were positively correlated with the pollen activity, and the seed setting rate was negatively correlated with the local precipitation.

Peonia, Feng dan’ (Paeonia ostia) do género Paeonia (o grupo lenhoso) é recomendado como uma nova cultura promissora na produção de óleos, devido aos elementos nutritivos na semente, no entanto, o desenvolvimento desta nova cultura está agora limitado devido às baixas taxas de fixação de sementes. Este estudo tem por objetivo identificar meios de melhorar a polinização cruzada de ‘Feng dan’ como o parental paterno, por ser uma cultura de óleo adequada, de modo a melhorar as taxas de fixação de sementes de ‘Feng dan’, e para prever a relação funcional entre o vigor do pólen e as taxas de fixação de sementes para melhorar a eficiência da experiência de polinização. Em primeiro lugar, foram realizados três passos para analisar o método de medição do vigor do pólen do pai paternal, que poderia ter uma relação forte com as taxas de fixação das sementes. Em segundo lugar, obter as taxas de fixação de sementes correspondentes por polinização cruzada. Em terceiro lugar, foi estabelecida a relação funcional entre dois índices que poderiam ser utilizados na prática da polinização cruzada. Os resultados indicaram que os melhores parentais paternos de ‘Feng dan’ eram cultivares de ‘Feng dan’ de outras populações de cultivo diferentes. A concentração de meio mais adequada para a germinação do pólen foi 100~150 g/L de sacarose, 0,06~0,08 g/L ácido bórico, e 50~200 g/L PEG-6000. Tanto a taxa de estabelecimento das sementes como o número de sementes foram positivamente correlacionados com a atividade do pólen, e a taxa de estabelecimento das sementes foi negativamente correlacionada com a precipitação local.

Cross pollination of different peony cultivars with 'Feng dan' / Polinização cruzada de diferentes cultivares de peônia com 'Fengdan'

Paeonia ostii ‘Feng dan is recommended as a new promising oil crop because of the nutrient elements in its seed; however, the development of this new oil crop is now limited due to its low seed setting rate. This study identified ways to improve the cross pollination of Feng dan as the maternal parent, which has been proved to be a suitable oil crop, so as to improve the seed setting rate of ‘Feng dan, and to predict the functional relationship between pollen vigor and seed setting rate. improving the efficiency of cross pollination. Three major steps were conducted, first, to screen the appropriate method of measuring pollen vigor of the paternal parent, which could have a strong relationship with seed setting rate. Second, to obtain the corresponding seed setting rate by cross pollination. Third, the functional relationship between two indexes was founded which could be used in cross pollination practice. Results indicated that the best paternal parents of ‘Feng dan were ‘Feng dan cultivars from other different cultivation populations; The most suitable medium concentration for pollen germination was 100~150 g/L sucrose, 0.06~0.08 g/L boric acid, and 50~200 g/L PEG-6000. Both the seed setting rate and the number of seed grains were positively correlated with the pollen activity, and the seed setting rate was negatively correlated with the local precipitation.(AU)

Peonia, Feng dan (Paeonia ostia) do género Paeonia (o grupo lenhoso) é recomendado como uma nova cultura promissora na produção de óleos, devido aos elementos nutritivos na semente, no entanto, o desenvolvimento desta nova cultura está agora limitado devido às baixas taxas de fixação de sementes. Este estudo tem por objetivo identificar meios de melhorar a polinização cruzada de ‘Feng dan como o parental paterno, por ser uma cultura de óleo adequada, de modo a melhorar as taxas de fixação de sementes de ‘Feng dan, e para prever a relação funcional entre o vigor do pólen e as taxas de fixação de sementes para melhorar a eficiência da experiência de polinização. Em primeiro lugar, foram realizados três passos para analisar o método de medição do vigor do pólen do pai paternal, que poderia ter uma relação forte com as taxas de fixação das sementes. Em segundo lugar, obter as taxas de fixação de sementes correspondentes por polinização cruzada. Em terceiro lugar, foi estabelecida a relação funcional entre dois índices que poderiam ser utilizados na prática da polinização cruzada. Os resultados indicaram que os melhores parentais paternos de ‘Feng dan eram cultivares de ‘Feng dan de outras populações de cultivo diferentes. A concentração de meio mais adequada para a germinação do pólen foi 100~150 g/L de sacarose, 0,06~0,08 g/L ácido bórico, e 50~200 g/L PEG-6000. Tanto a taxa de estabelecimento das sementes como o número de sementes foram positivamente correlacionados com a atividade do pólen, e a taxa de estabelecimento das sementes foi negativamente correlacionada com a precipitação local.(AU)

Cross pollination of different peony cultivars with 'Feng dan' / Polinização cruzada de diferentes cultivares de peônia com 'Fengdan'

ABSTRACT: Paeonia ostii 'Feng dan' is recommended as a new promising oil crop because of the nutrient elements in its seed; however, the development of this new oil crop is now limited due to its low seed setting rate. This study identified ways to improve the cross pollination of Feng dan as the maternal parent, which has been proved to be a suitable oil crop, so as to improve the seed setting rate of 'Feng dan', and to predict the functional relationship between pollen vigor and seed setting rate. improving the efficiency of cross pollination. Three major steps were conducted, first, to screen the appropriate method of measuring pollen vigor of the paternal parent, which could have a strong relationship with seed setting rate. Second, to obtain the corresponding seed setting rate by cross pollination. Third, the functional relationship between two indexes was founded which could be used in cross pollination practice. Results indicated that the best paternal parents of 'Feng dan' were 'Feng dan' cultivars from other different cultivation populations; The most suitable medium concentration for pollen germination was 100~150 g/L sucrose, 0.06~0.08 g/L boric acid, and 50~200 g/L PEG-6000. Both the seed setting rate and the number of seed grains were positively correlated with the pollen activity, and the seed setting rate was negatively correlated with the local precipitation.

RESUMO: Peonia, Feng dan' (Paeonia ostia) do género Paeonia (o grupo lenhoso) é recomendado como uma nova cultura promissora na produção de óleos, devido aos elementos nutritivos na semente, no entanto, o desenvolvimento desta nova cultura está agora limitado devido às baixas taxas de fixação de sementes. Este estudo tem por objetivo identificar meios de melhorar a polinização cruzada de 'Feng dan' como o parental paterno, por ser uma cultura de óleo adequada, de modo a melhorar as taxas de fixação de sementes de 'Feng dan', e para prever a relação funcional entre o vigor do pólen e as taxas de fixação de sementes para melhorar a eficiência da experiência de polinização. Em primeiro lugar, foram realizados três passos para analisar o método de medição do vigor do pólen do pai paternal, que poderia ter uma relação forte com as taxas de fixação das sementes. Em segundo lugar, obter as taxas de fixação de sementes correspondentes por polinização cruzada. Em terceiro lugar, foi estabelecida a relação funcional entre dois índices que poderiam ser utilizados na prática da polinização cruzada. Os resultados indicaram que os melhores parentais paternos de 'Feng dan' eram cultivares de 'Feng dan' de outras populações de cultivo diferentes. A concentração de meio mais adequada para a germinação do pólen foi 100~150 g/L de sacarose, 0,06~0,08 g/L ácido bórico, e 50~200 g/L PEG-6000. Tanto a taxa de estabelecimento das sementes como o número de sementes foram positivamente correlacionados com a atividade do pólen, e a taxa de estabelecimento das sementes foi negativamente correlacionada com a precipitação local.

Effect of heterospecific pollen deposition on pollen tube growth depends on the phylogenetic relatedness between donor and recipient.

Co-flowering plant species may interact via pollinators leading to heterospecific pollen transfer with consequences for plant reproduction. What determines the severity of heterospecific pollen effect on conspecific pollen performance is unclear, but it may depend on the phylogenetic relatedness of the interactors (pollen donors and recipient). The heterospecific pollen effect might also depend on the extent to which plants are exposed to heterospecific pollen over ecological or evolutionary timescales. For instance, generalist-pollinated plant species might tolerate heterospecific pollen more than specialists. Here, we tested whether heterospecific pollen effects are stronger between closely related species than phylogenetically distant ones in a tropical highland community. Then, based on these results, we determined whether responses to heterospecific pollen were stronger in generalized vs. specialized plant species. We applied heterospecific pollen from close (congeneric) or distant (different families) donors alone or with conspecific pollen on stigmas of three recipient species (one generalist, Sisyrinchium wettsteinii; and two specialists, Fuchsia campos-portoi and Fuchsia regia) and scored pollen tube performance in styles. In all species, pollen from closely related donors grew pollen tubes to the base of the style indicating a high potential to interfere with seed set. Conversely, distantly related heterospecific pollen had no effect on either specialist Fuchsia species, whereas enhanced performance of conspecific pollen was observed in generalist S. wettsteinii. The strong effect of phylogenetic relatedness of donor and recipient might have obscured the role of pollination specialization, at least for the three species examined here. Therefore, phylogenetic relatedness mediated the effect of heterospecific pollen on post-pollination success, with possible consequences for reproductive trait evolution and community assembly for further studies to explore.

Storage of orchid pollinia with varying lipid thermal fingerprints.

Orchid pollinia have the potential to make a valuable contribution to current techniques of germplasm storage and assisted reproduction, yet information regarding their preservation and their ability to remain viable over time is currently limited. Dactylorhiza fuchsii and Disa uniflora were used as models for investigating potential techniques for storing orchid pollinia. Initially, freshly harvested pollinia of Dact. fuchsii were incubated at 25 °C and 100% RH (relative humidity) for up to 7 days and germinated in vitro. For pollinia from both species, moisture sorption isotherms were constructed and thermal fingerprints generated using differential scanning calorimetry (DSC). Pollinia were stored at three temperatures (5, - 18 and - 196 °C) after equilibration at four different RHs (5, 33, 50 and 75%) and germinated. The isotherms and DSC results varied between species. Compared with D. uniflora, pollinia of Dact. fuchsii consistently equilibrated at higher moisture content (MC) for each RH, had less detectable lipids by DSC and had shorter lifespans, remaining viable after 3-4 months only at - 20 and - 196 °C and at low RH (5 and 33%). Both species' pollinia stored well at - 20 °C and - 196 °C, although there was some evidence of a small loss of viability under cryopreservation. In conclusion, pollen of these two species can be stored successfully for at least 3-4 months, and to maximize the pre-storage quality, it is recommended that fresh pollen is collected from flowers just prior to anthesis.

Pollen morphology and viability in Bromeliaceae

ABSTRACT Pollen morphology characterization is important in taxonomy, conservation and plant breeding, and pollen viability studies can support breeding programs. This study investigated pollen morphology and male fertility in 18 species of Bromeliaceae with ornamental potential. For morphological characterization, pollen grains were acetolyzed and characterization of exine was done using scanning and transmission electron microscopy. Pollen viability was investigated by in vitro germination and histochemical tests. Species belonging to Aechmea and Ananas genera presented medium size pollen, except for Ae. fasciata, with large pollen. Al. nahoumii, P. sagenarius and the Vriesea species analyzed showed large pollen, except for V. carinata, with very large pollen. Pollen of Aechmea, Ananas and P. sagenarius presented bilateral symmetry, diporate, exine varying from tectate to semitectate. Al. nahoumii and Vriesea species presented pollen with bilateral symmetry, monocolpate; exine was semitectate, reticulate and heterobrochate. Germination percentage and tube growth were greater in SM and BKM media. Histochemical tests showed pollen viability above 70% for all species, except for Ananas sp. (40%). Pollen morphology is important for the identification of species, especially in this family, which contains a large number of species. High rates of viability favor fertilization and seed production, essential for efficient hybrid production and conservation.

Pollen Aquaporins: The Solute Factor.

In the recent years, the biophysical properties and presumed physiological role of aquaporins (AQPs) have been expanded to specialized cells where water and solute exchange are crucial traits. Complex but unique processes such as stomatal movement or pollen hydration and germination have been addressed not only by identifying the specific AQP involved but also by studying how these proteins integrate and coordinate cellular activities and functions. In this review, we referred specifically to pollen-specific AQPs and analyzed what has been assumed in terms of transport properties and what has been found in terms of their physiological role. Unlike that in many other cells, the AQP machinery in mature pollen lacks plasma membrane intrinsic proteins, which are extensively studied for their high water capacity exchange. Instead, a variety of TIPs and NIPs are expressed in pollen. These findings have altered the initial understanding of AQPs and water exchange to consider specific and diverse solutes that might be critical to sustaining pollen's success. The spatial and temporal distribution of the pollen AQPs also reflects a regulatory mechanism that allowing a properly adjusting water and solute exchange.

The effect of three plant bioregulators on pollen germination, pollen tube growth and fruit set in almond [Prunus dulcis (Mill.) D.A. Webb] cvs. Non Pareil and Carmel

Background In commercial almond [Prunus dulcis (Mill.) D.A. Webb] orchards in Chile, the percentage of fruit set is low and commonly reaches 5–30%. As almond trees bloom during a cool period and also suffer from self-incompatibility, any factor that can improve pollination, pollen germination and pollen tube growth or extend the effective pollination period, such as the application of plant bioregulators (PBRs), should be beneficial for fruit production. Results Three plant bioregulators (PBRs): brassinolide (BL), gibberellic acid (GA3) and kinetin (KN) were evaluated for pollen germination and pollen tube growth in vitro, as well as for fruit set in almond cultivars Non Pareil and Carmel, in central Chile, during the 2013 and 2014 seasons. For pollen germination in vitro, the BL concentration of 10 mg L- 1 had the highest value in Non Pareil (97.7%), after 4 h germination in 2014 growing season (the control was 90.9%). KN at a concentration of 50 µL L- 1 induced the longest pollen tube growth of 1243.4 µm in Carmel after 8 h germination in 2013 (the control was 917.7 µm). In Non Pareil, the highest percentage of fruit set (31.0%) was achieved in 2014 by spraying during bloom at pink bud stage with KN at 50 µL L- 1 (the control was 16.7%). Conclusions A significant favorable effect of the tested PBRs was observed in pollen germination and pollen tube growth in vitro, as well as on fruit set in Non Pareil and Carmel almonds.

Viabilidade dos grãos de pólen de flores de pinheira (Annona squamosa) em diferentes horários / Viability of the sugar apple (Annona squamosa) pollen grains at different hours of the day

No manejo do cultivo da pinha (Annona squamosa), a polinização artificial é uma prática preconizada para, obter maior pegamento dos frutos bem como uniformização do formato dos mesmos. Nesse sentido, conduziu-se este trabalho, com o objetivo de avaliar a viabilidade dos grãos de pólen de flores de pinheira em diferentes horários de coleta. O pólen foi obtido a partir de flores no estádio funcionalmente estaminada. Foram avaliados oito horários de coleta de pólen: zero hora, 1 hora, 2 horas, 3 horas, 4 horas, 5 horas, 6 horas e 7 horas da manhã. Foi utilizado meio de cultura padrão para germinação de pólen, com concentração de 10% de sacarose. As flores foram coletadas nos horários estabelecidos e os grãos de pólen foram retirados das anteras com auxílio de um pincel número 2 e em seguida inoculados em placas de Petri contendo o meio de cultura. O delineamento experimental foi inteiramente casualizado com quatro repetições, sendo cada parcela experimental constituída por duas placas Petri. Foram contados 100 grãos de pólen por placa. Após 6 horas de inoculação, os grãos de pólen foram visualizados sob lupa. Foram considerados germinados os grãos de pólen que possuíam tubo polínico com tamanho igual ou superior ao diâmetro do próprio pólen. Não foram observadas diferenças significativas entre o horário de coleta dos grãos de pólen. A percentagem média da germinação variou de 46,75% a 53,62% dos grãos de pólen germinados.

In the management of the sugar apple (Annona squamosa) crop, the artificial pollination is a preconized practice to obtain higher establishment of the fruits as well as their standardization. This study was carried out to evaluate the viability of sugar apple pollen grains at different collecting times. The pollens were obtained from the flowers at the functional staminate stage. The pollen grains were collected every hour, starting from 00:00am and ending at 07:00am, totalizing 8 collections. The standard culture medium with 10 percent sucrose was used for the germination of the pollen. The flowers were collected at the scheduled hours and the pollen grains were taken from the anthers, by using a brush # 2. They were then inoculated on Petri dishes containing the culture medium. The completely randomized experimental design was used with four replicates, and each plot was constituted by two Petri dishes. One hundred pollen grains were counted in each Petri dish. After six hours of inoculation, the pollen grains were visualized under magnifying glass. Those pollen grains containing the pollinic tube with the same or higher size than their own pollen diameter were considered germinated. No significant differences were found among the different hours of the pollen grain collections, and the germination percentage ranged from 46.75% to 53.62%.