GAMYB transcription factor LoMYB65 from lily plays a vital role in pollen development

Lily(Lilium spp.)is an important horticultural crop,but its use is limited due to serious pollen contamination problems.There are many studies on pollen development in model plants,but few on flower crops such as lilies.Gibberellin(GA)is a large class of hormones and plays an important role in plant vegetative growth and reproductive development.GAMYB is a group of the R2R3-MYB family upregulated by gibberellin,and plays an important role in anther development.Here,we isolated a novel GAMYB,named LoMYB65,from lily,which was closely related to the AtMYB65 and AtMYB33 in Arabidopsis.Fluorescence quantitative PCR results showed that LoMYB65 was mainly expressed in lily anthers.LoMYB65 could be activated by 288μmol·L^(-1)GA3treatment and the LoMYB65 protein was located in the nucleus and cytoplasm,and had transactivation in yeast and tobacco leaf cells.The conserved motif within 226 amino acids of the C-terminal of LoMYB65 contributed to its transactivation.Overexpression of LoMYB65 caused dwarf phenotype,unnormal tapetum development,less seeds of siliques in transgenic Arabidopsis plants,the transgenic plants showed partly male sterile.Simultaneously,silencing of LoMYB65 with VIGS(Virus Induced Gene Silencing)in lily anthers caused unnormal pollen development and reduced the pollen amount.Overexpression of LoMYB65 in Arabidopsis and silencing of LoMYB65 in lily resulted in decreased pollen counts,so we speculate that LoMYB65 may be dose-dependent.Overall,these findings suggest that LoMYB65 may play an important role in anther development and pollen formation in lily.LoMYB65 may provide a useful candidate gene for pollenless breeding of lily.

Effect of different drying methods on the amino acids,α-dicarbonyls and volatile compounds of rape bee pollen

The significant demand for high quality food has motivated us to adopt appropriate processing methods to improve the food nutritional quality and flavors.In this study,the effects of five drying methods,namely,pulsed vacuum drying(PVD),freeze drying(FD),infrared drying(IRD),hot-air drying(HAD)and sun drying(SD)on free amino acids(FAAs),α-dicarbonyl compounds(α-DCs)and volatile compounds(VOCs)in rape bee pollen(RBP)were determined.The results showed that FD significantly released the essential amino acids(EAAs)compared with fresh samples while SD caused the highest loss.Glucosone was the dominantα-DCs in RBP and the highest loss was observed after PVD.Aldehydes were the dominant volatiles of RBP and SD samples contained more new volatile substances(especially aldehydes)than the other four drying methods.Comprehensively,FD and PVD would be potential methods to effectively reduce the quality deterioration of RBP in the drying process.

Effect of wall-disruption on nutrient composition and in vitro digestion of camellia and lotus bee pollens

The nutrient digestion,absorption and biological activity of bee pollen may be limited due to the complex pollen wall.Here,the effect of superfine grinding technology on the release of nutrients from bee pollen were investigated,and their antioxidant activities and in vitro digestion were explored in this study.Results showed that the content of nutrients in bee pollen increased after wall disruption.Among them,fat content increased by 22.55%-8.31%,protein content increased by 0.54%-4.91%,starch content increased by 36.31%-48.64%,soluble sugar content increased by 20.57%-29.67%,total phenolic acid content increased by 11.73%-86.98%and total flavonoids content increased by 14.29%-24.79%.At the same time,the antioxidant activity increased by 14.84%-46.00%.Furthermore,the active components such as phenolic compounds in the wall-disruption bee pollen were more readily to be released during the in vitro digestion,and easier to be absorbed because of their higher bioaccessibility.Antioxidant activities during in vitro digestion were also improved in walldisruption bee pollen.These findings provide evidence that bee pollen wall disruption was suggested,thus,it is more conducive to exerting the value of bee pollen in functional foods.

Enhancer of Shoot Regeneration 2(ESR2)regulates pollen maturation and vitality in watermelon(Citrullus lanatus)

Watermelon(Citrullus lanatus)holds global significance as a fruit with high economic and nutritional value.Exploring the regulatory network of watermelon male reproductive development is crucial for developing male sterile materials and facilitating cross-breeding.Despite its importance,there is a lack of research on the regulation mechanism of male reproductive development in watermelon.In this study,we identified that ClESR2,a VIIIb subclass member in the APETALA2/Ethylene Responsive Factor(AP2/ERF)superfamily,was a key factor in pollen development.RNA insitu hybridization confirmed significantClESR2 expression in the tapetum and pollen during the later stage of anther development.The pollens of transgenic plants showed major defects in morphology and vitality at the late development stage.The RNA-seq and protein interaction assay confirmed that ClESR2 regulates pollen morphology and fertility by interacting with key genes involved in pollen development at both transcriptional and protein levels.These suggest that Enhancer of Shoot Regeneration 2(ESR2)plays an important role in pollen maturation and vitality.This study helps understand the male reproductive development of watermelon,providing a theoretical foundation for developing male sterile materials.

High temperature treatment induced production of unreduced 2n pollen in Camellia oleifera

Unreduced gametes through chromosome doubling play a major role in the process of plant polyploidization.Our previous work confirmed that Camellia oleifera can produce natural 2n pollen,and it is possible to induce the 2n pollen formation by high temperature treatment.This study focused on the optimization of the 2n pollen induction technique and the mechanisms of high temperature-induced2n pollen formation in C.oleifera.We found that the optimal protocol for inducing 2n pollen via high temperature was to perform 45℃with4 h at the prophaseⅠstage of the pollen mother cells(PMCs).Meanwhile,high temperature significantly decreased the yield and fertility of2n pollen.Through the observation of meiosis,abnormal chromosome and cytological behaviour was discovered under high-temperature treatment,and we confirmed that the formation of 2n pollen is caused by abnormal cell plate.Based on weighted gene co-expression network analysis,fifteen hub genes related to cell cycle control were identified.After male flower buds were exposed to heat shock,polygalacturonase gene(CoPGX3)was significantly upregulated.We inferred that high temperature causes the CoPGX3 gene to be overexpressed and that CoPGX3 is redistributed into the cytosol where it degrades cytoplasmic pectin,which leads to an abnormal cell plate.Furthermore,abnormal cytokinesis resulted in the formation of dyads and triads,and PMCs divided to produce 2n pollen.Our findings provide new insights into the mechanism of 2n pollen induced by high temperature in a woody plant and lay a foundation for further ploidy breeding of C.oleifera.

Modern pollen-vegetation relationships along altitudinal vegetation belts in the Leigong Mountain of southwestern China

Understanding the distribution,dispersal,and correlation of modern pollen with vegetation in mountainous regions is essential for establishing accurate modern analogs for fossil pollen records.This study,conducted in Leigong Mountain on the YunnanGuizhou Plateau of southwestern China,involved the collection of 35 surface soil samples from diverse vegetation communities along an elevational gradient ranging from 1210 to 1875 meters.The results reveal a close correspondence between modern pollen assemblages and vegetation zones.Principal Component Analysis(PCA)results indicate that pollen assemblages can effectively distinguish between subtropical montane evergreen broad-leaved forest(SEBF)and subtropical montane deciduous broadleaved forest(SDBF).However,both SEBF and SDBF show significant overlap with subtropical montane evergreen-deciduous broad-leaved mixed forest(SEMF).Detrended Correspondence Analysis(DCA)results clearly distinguish the three vegetation zones,and the first axis of DCA shows a significant positive correlation with elevation(p<0.01,R=0.48).Discriminant Analysis(DA)successfully assigns 94.4%of the modern pollen samples to their respective vegetation zones.Pollen taxa such as Impatiens,Astertype,and Rosaceae exhibit significant indicative capabilities for the SEBF zone,effectively distinguishing this vegetation zone from others.Pinus and Alnus display overrepresentation in the Leigong Mountain region,while Quercus(D,deciduous-type)and Poaceae exhibit high representation in the SEBF zone.In the SEBF zone,both pollen diversity and richness are the lowest.Our study reveals the complex relationship between the richness and diversity of pollen and vegetation.The diversity and richness of tree and shrub pollen are found to be lower than those of the corresponding plants.The pollen-vegetation relationship elucidated in this study serves as a critical reference for reconstructing ancient environments from fossil pollen retrieved in this region.

Increased sugar content impairs pollen fertility and reduces seed-setting in high-photosynthetic-efficiency rice

Crop yield depends on biomass,which is primarily associated with photosynthesis.We previously demonstrated that two photorespiratory bypasses,i.e.,GOC(glycolate oxidase+oxalate oxidase+catalase)and GCGT(glycolate oxidase+catalase+glyoxylate carboligase+tartronic semialdehyde reductase),significantly increased photosynthesis,biomass,and grain yield,but decreased seed-setting rates in rice.This study explored the underlying mechanism of how elevated photosynthetic efficiency impacted the seed-setting.First,pollen germination assessed in vivo and in vitro,revealed a reduced germination rate in GCGT rice.Subsequent analysis found that photosynthates highly accumulated in the leaves and stems;sucrose and soluble sugar levels were increased but the starch level was reduced in the anthers.Uridine diphosphate glucose(UDP-Glc)was increased but uridine diphosphate galactose(UDP-Gal)was unaltered,thus causing an imbalance in the UDP-Glc/UDP-Gal ratio in GCGT anthers.Most anthers in GCGT plants had two locules in contrast to four in the wild-type(WT).Pollen tapetum was developmentally abnormal,and genes related to sucrose synthesis,transport,and tapetal programmed cell death(PCD)were upregulated,whereas those involved in starch synthesis and conversion were downregulated in GCGT anthers.Taken together,our results demonstrated that an increase in sugar content was the primary factor causing reduced seed-setting rates in high photosynthetic efficiency rice,during which metabolic disorder of sugars and UDP sugar imbalance in anthers lead to impaired pollen fertility.

Comparative Study on Floral Organs and Pollen Characteristics of Different Varieties of Sorrel

The floral organ morphology,pollen morphology,quantity,and viability of Rumex,Rumex hanus F1,Rumex hanus 1,and Rumex hanus 2 were compared and analyzed.The results showed that the longitudinal diameter of petals was not significantly different among different varieties of sorrel,and the transverse diameter and corolla diameter showed differences to varying degrees.They all had six stamens and three stigmas.The pollen shape of the four varieties was prolate,and the polar view was trilobated and round.They all had three germinal furrows,and the outer wall of the pollen was decorated with small cavities and spiny patterns,with warty protrusions.The equatorial axis length of pollen of different varieties of sorrel was significantly different(P<0.05).There were no significant differences in pollen amount among different varieties.There were six anthers in a single flower,featuring anther dehiscence.The pollen viability of Rumex hanus 1 was significantly lower than that of the other three varieties(P<0.05).Therefore,through the analysis of the floral organs and morphological characteristics of anthers of sorrel,the varieties of sorrel can be effectively distinguished and identified.

Morphological and cytological assessments reveal pollen degradation causes pollen abortion in cotton cytoplasmic male sterility lines

Background Understanding the mechanism of male sterility is crucial for producing hybrid seeds and developing sterile germplasm resources.However,only a few cytoplasmic male sterility(CMS)lines of cotton have been produced due to several challenges,like inadequate variation of agronomic traits,incomplete sterility,weak resilience of restorer lines,and difficulty in combining strong dominance.Therefore,the morphological and cytological identification of CMS in cotton will facilitate hybrid breeding.Results Two F_(2) segregating populations of cotton were constructed from cytoplasmic male sterile lines(HaA and 01A,maternal)and restorer lines(HaR and 26R,paternal).Genetic analysis of these populations revealed a segregation ratio of 3:1 for fertile to sterile plants.Phenotypic analysis indicated no significant differences in traits of flower bud development between sterile and fertile plants.However,sterile plants exhibited smaller floral organs,shortened filament lengths,and anther atrophy on the flowering day in comparison with the fertile plants.When performed scanning electron microscopy(SEM),the two F_(2) populations revealed morphological variations in the anther epidermis.Cellular analysis showed no significant differences in pollen development before pollen maturation.Interestingly,between the pollen maturation and flowering stages,the tapetum layer of sterile plants degenerated prematurely,resulting in abnormal pollen grains and gradual pollen degradation.Conclusion The results of this study suggest that fertility-restoring genes are controlled by a single dominant gene.Sterile plants exhibit distinctive floral morphology,which is characterized by stamen atrophy and abnormal anthers.Pollen abortion occurs between pollen maturity and flowering,indicating that premature tapetum degradation may be the primary cause of pollen abortion.Overall,our study provides a theoretical basis for utilizing CMS in hybrid breeding and in-depth investigation of the dominant configuration of cotton hybrid combinations,mechanisms of sterility,and the role of sterile and restorer genes.

Treatments of Pollen with Different Solvents Affect the Seed Setting Rate of Rice(Oryza sativa L.)

This study aimed to investigate the effects of treatments of rice pollen with different solvents on the seed setting rate.Rice pollen was treated with ultrapure water,0.5%dimethyl sulfoxide,ethanol(1%,5%,and 8%),and 0.5%dimethyl sulfoxide+8%ethanol,and the pollen without treatment was taken as the control.Then,crossing was conducted with the treated rice plants as the male parents and plants subjected to emasculation by warm water as the female parents.The seed setting rates of the male and female parents were then recorded.The results showed that the treatments with different solvents significantly decreased the seed setting rates of both male and female parents.The treatment with 1%ethanol resulted in the highest seed setting rates for both male and female parents,while the treatment with 0.5%dimethyl sulfoxide resulted in the lowest seed setting rates for both parents.Although these solvents affected the seed setting rate of rice,they served as intermediate solvents to dissolve chemicals used in the induction of rice haploids.This study layed a technical foundation for subsequent chemical induction of rice haploids.

Allergen degradation of bee pollen by lactic acid bacteria fermentation and its alleviatory effects on allergic reactions in BALB/c mice

Food allergy as a global health problem threatens food industry.Bee pollen(BP)is a typical food with allergenic potentials,although it performs various nutritional/pharmacological functions to humans.In this study,lactic acid bacteria(LAB)were used to ferment Brassica napus BP for alleviating its allergenicity.Four novel allergens(glutaredoxin,oleosin-B2,catalase and lipase)were identified with significant decreases in LAB-fermented BP(FBP)than natural BP by proteomics.Meanwhile,metabolomics analysis showed significant increases of 28 characteristic oligopeptides and amino acids in FBP versus BP,indicating the degradation of LAB on allergens.Moreover,FBP showed alleviatory effects in BALB/c mice,which relieved pathological symptoms and lowered production of allergic mediators.Microbial high-throughput sequencing analysis showed that FBP could regulate gut microbiota and metabolism to strengthen immunity,which were closely correlated with the alleviation of allergic reactivity.These findings could contribute to the development and utilization of hypoallergenic BP products.

DIA-based proteome profiling with PRM verification reveals the involvement of ER-associated protein processing in pollen abortion in Ogura CMS cabbage

Ogura cytoplasmic male sterility(Ogura CMS)is extensively applied in hybrid seed production in cruciferous crops.However,the posttranscriptional molecular basis of Ogura CMS in cruciferous crops remains elusive.Here,a data-independent acquisition-based proteomic approach coupled with a parallel reaction monitoring-based targeted proteomic assay was used to analyze the proteome dynamics of Ogura CMS cabbage line RM and its maintainer line RF during floral bud development to obtain insights into the mechanism underlying Ogura CMS in cruciferous crops.A total of 9162 proteins corresponding to 61464 peptides were identified in RM and RF floral buds.The proteomic fluctuation of RM was weaker than that of RF.Differences in protein expression between RM and RF gradually enlarged with floral bud development.Fifteen continually up-regulated and eight continually down-regulated proteins were found in RM relative to RF throughout floral bud development.Differentially expressed proteins between RM and RF during floral bud development were implicated in the endoplasmic reticulum(ER)-associated protein processing pathway,in which most of them exhibited down-regulated expression in RM.These data suggest that ER-associated protein processing may be involved in pollen abortion in Ogura CMS cabbage by inhibiting the expression of critical factors.Our findings not only deepen the understanding of the molecular mechanisms of Ogura CMS in cruciferous crops but also provide better guidance for applying Ogura CMS in the hybrid breeding of cruciferous crops.

Effects of Low Energy Argon Ion Implantation on Maize Pollen Germination and the Cytosolic Ca^(2+) Distribution during Pollen Germination Process

To investigate the effect of low energy ion implantation on maize pollen germination and cytosolic Ca2+ distribution during pollen germination process, the argon ion (Ar+) with energy of 30 keV, dose of 0.78 ×1015-13×1015 ion/cm2 was implanted into maize pollen by irradiation, and the germination of pollen and cytosolic Ca2+ distribution during pollen germination process of the Ar+ implanted pollen were studied. The results showed that when been irradiated with Ar+ with dose of 5.2×1015 ion/cm2, the germination rate of maize pollen increased remarkably, while implantation of ions with dose exceeding 5.2×1015 ion/cm2 sharply decreased the germination rate of maize pollen. Meanwhile, tracing of esterified Ca2+ fluorescence probe fluo-3 AM for intact pollen showed that variation of cytosolic Ca2+ concentration was consistent with the change of pollen fertility. The dynamics of cytosolic Ca2+ concentration caused by low energy ion implantation may be concluded as the initial effects of pollen germination.

Influence of Plant Growth Regulators on Pollen Germination and Growth of Xinjiang Apricots

[Objective] This study aimed to investigate the influence of different plant growth regulators on apricot pollen germination and pollen tube growth. [Method] Pollens of six kinds of Xinjiang apricots were cultured in solid media supplemented with five plant growth regulators （GA3 , NAA, 2, 4-D, 6-BA, IAA）. Then the rate of pollen germination and the length of pollen tube were respectively measured. [Result] In a certain concentration range, GA3 most significantly promoted the pollen germination and the pollen tube growth of Shushanggan, Kalayulvke, Dayoujia, Yiliakeyulvke and Kabakehuanna; NNA had the strongest improvement function on Kumaiti’s pollen germination and pollen tube growth. [Conclusion] All the five plant growth regulators promoted the pollen germination and the pollen tube growth of apricots at low concentration but inhibited them at high concentration.

Vitality and storage condition of Syringa pollen

The fresh pollen vitality, the effect of different storage conditions on the pollen vitality, and the difference of vitality among the species of seven species of Syringa were determined in Shenyang, China. The results indicated that the pollen vi-tality (81.5%) of Syringa villosa was the highest among the seven tested species, followed by S. microphylla and S. meyeri, and that of S. oblata var. affinis was the lowest. The low temperature was the best condition for storage of pollen of Syringa, and the most proper temperature for the storage was 0-2 癈. The storability of S. microphylla was the best of all, and it could be stored over 60 days at the temperature of 0-2 癈, next was S. villosa and S. meyeri.

Mapping of S-b Locus for F_1 Pollen Sterility in Cultivated Rice Using PCR Based Markers

In cultivated rice ( Oryza sativa L.), F-1 pollen sterility is controlled by at least 6 loci of the F, pollen sterility genes. To map S-b, one of loci, rice variety Taichung 65 (T65) carrying S-b(j)/S-b(j) and its near-isogenic line TIST2 carrying S-b(i)/S-b(i) were used to develop the mapping population. One hundred and fifty-eight microsatellite markers were selected to survey T65 and TISL2. RM13 on chromosome 5 was found to be polymorphic between them. Cosegregation indicated that RM13 was closely linked with locus S-b. Eleven RFLP markers were selected on the corresponding region from the genetic map of Rice Genome Research Program (RGP) of Japan to convert into sequence-tagged site (STS) markers. Amplicon length polymorphism (ALP) was carried out, but none of them was found to be polymorphic between T65 and TISL2. Then PCR-based RFLP (PBR) was done using six 4-nucleotide recognizing restriction endonucleases. Polymorphism was detected when PCR products of R830STS and R2213SSTS were digested with Taq I. Genetic analysis indicated that the distance between locus S-b and markers, R830STS, RM13 and R2213SSTS were 3.3 cM (centi-Morgan), 5.2 cM and 5.5 cM, respectively. These PCR-based markers could be directly used in marker-assisted selection. The technical system combining genetic mapping and PCR-based marker-assisted selection will facilitate the development of molecular breeding.

Observation Study on Pollen Morphology of Chinese Pennisetum

[Objective] The aim was to study pollen morphology of Pennisetum native species.[Method] We carried out the observation by optical microscope and scanning electron microscope on six materials of pollen grains including three species and three transmogrifications from one variant of Pennisetum native species.[Result] The pollen grains were globose or subglobose.Germination aperture which located at distal side was singular and its circumambience was thickened.Pollen wall which had two layers was thin and easy to be crimpled.There were obvious differences between ektexine ornamentations as well as particle sizes.[Conclusion] The question of low seed-setting rate of Pennisetum native species had relations with the withered pollen morphology to some extent.The differences between pollen morphology as well as ektexine ornamentations accorded with the division of species and variants from Pennisetum.In addition,the relationship between pollen morphology and seed-setting rate provided references to introduce good grass seed and choose breeding parent materials of Pennisetum.

Scanning Electron Microscope Observation of Three Species on Pollen Grain Morphology and Leaf Epiderm Shape in Gramineae Triticeae

The pollen grain morphology and leaf epiderm shape and microstructural of Rye（ Secale cereal ）, common wheat and Octoploid Trititrigia were observed with the scanning electron microscope（SEM）. The results revealed that there were great differences between pollen and leaf epiderm microcosmic morphology in three species. Such characteristics of Octoploid tnticalwere between those of common wheat and Rye. The morphology of pollen grain and leaf epiderm shape counld be used as an index for taxonomy for genera and species, and had somewhat scientif- ic references to identify new multiploid species created by chromosome engineering.

Pollen Assemblage Features of Modern Water Samples from the Shiyang River Drainage, Arid Region of China

Pollen analysis of 30 modem water samples from the Shivang River, an internal river system located between the Tengger and Badain Jaran deserts, Northwest China was carried out to examine the river's capacity to carry pollen and spores, and to assess the contribution of the water-borne pollen to pollen assemblages in lake sediments at the end of the river system. Results indicate the pollen assemblages in water samples consist of both local and upland pollen. Percentages of upland pollen reach 30% - 60%, and pollen assemblages in water samples do not indicate the nature of local vegetation at the sampling sites. Fluvial currents have the capacity to transport large quantities of pollen long distances, and the contribution of this fluvial transported pollen is relatively high, For example, percentages of Picea Dietr. pollen in water samples at sampling sites 130 km and 145 km away from Picea forests reach 16.5% and 7.7%, respectively. Fluvial pollen transport occurs primarily during flood periods, and pollen concentrations from the flood samples are 17.1 - 12.5 times those from normal fluvial flow. Reservoirs affect pollen transportation since pollen is deposited at reservoir inlets and pollen concentrations are much reduced at reservoir outlets. Human activity can thus change natural features of pollen transportation and deposition. The main factors influencing pollen concentrations and assemblages are sampling time, sampling location, and rainfall intensity.

Ultrastructural Observation on the Intra-and Intercellular Microtrabecular Network of the Pollen Mother Cells in Onion ( Allium cepa )

Using DGD embedment_free electron microscopy, ultrastructural observation on the intra_ and intercellular microtrabecular network (MN) of the pollen mother cells (PMC) of the whole meiotic prophase Ⅰ in onion ( Allium cepa L.) was performed. Complex nuclear MN was observed in the nucleus of PMCs, spreading throughout the nuclear region. The nucleolus and chromosomes were connected with the MN filament network. The uniformity of nuclear MN changed with the development of the PMCs. A lamina_like structure surrounded the nucleus and joined the MN in nucleus and in cytoplasm, but it disassembled at the end of prophase Ⅰ. There was also a complex cytoplasmic MN in PMCs, without obvious variation during the prophase Ⅰ. Furthermore, MN in cytoplasmic connections (plasmodesmata and cytoplasmic channels) was noticed to link the frameworks in two neighboring PMCs into one entity. Cytomixis was observed at synizesis of prophase Ⅰ in this experiment, and MN in cytoplasm and in nucleus was noticed to distribute in these granules which migrated from one PMC into its neighboring cell. At this time the nucleus moved aside from center of the PMC, but the rest of the cell was still fulfilled with MN filaments. The relationships of nuclear MN with nucleolus and chromosomes, lamina with nucleus, as well as intra_ and intercellular MN with cytomixis are discussed in this paper.