Pollen Stage Research Articles

Histochemical and ultrastructural analysis of tapetum and sporoderm development in relation to precocious pollenkitt production of Garcinia dulcis (Roxb.) Kurz.

Garcinia dulcis (Roxb.) Kurz (Clusiaceae) is a medicinal plant native to Southeastern Asia, with a peculiar, precocious pollenkitt production in early microspore development. We aimed to find out whether different secretory activities of the tapetum or a premature sporoderm development provides additional evidence for our recent hypothesis for the precocious pollenkitt production. Histology, histochemistry and ultrastructure of tapetum and sporoderm development during pollenkitt secretion in Garcinia dulcis were conducted, based on light and electron microscopy analysis. The results showed that Garcinia dulcis possesses normal pollen development. The presence of two different pollen coating types, precocious pollenkitt (L1) and common pollenkitt (L2), in the anther tapetum indicate that they are produced in two different active stages of the secretory tapetum. The precocious pollenkitt production and transport to the locule takes place in early active tapetal cells at early tetrad to early microspore stage and is ongoing until late microspore stage. The production of the second type of pollenkitt (L2) starts shortly after the first active tapetum stage together with the formation of sporopollenin precursors. The sporoderm formation was completed at late microspore stage, when the tapetal cell walls start to disintegrate. Orbicules are lining the inner tapetum wall at middle to late microspore stage. ER (during early microspore stage) and plastids (during late microspore stage) were the two main sources of pollenkitt, which finally fused to pollenkitt droplets when the tapetal cells degenerated at mature bicellular pollen stage.

Functional Characterization of CsSWEET5a, a Cucumber Hexose Transporter That Mediates the Hexose Supply for Pollen Development and Rescues Male Fertility in Arabidopsis.

Pollen cells require large amounts of sugars from the anther to support their development, which is critical for plant sexual reproduction and crop yield. Sugars Will Eventually be Exported Transporters (SWEETs) have been shown to play an important role in the apoplasmic unloading of sugars from anther tissues into symplasmically isolated developing pollen cells and thereby affect the sugar supply for pollen development. However, among the 17 CsSWEET genes identified in the cucumber (Cucumis sativus L.) genome, the CsSWEET gene involved in this process has not been identified. Here, a member of the SWEET gene family, CsSWEET5a, was identified and characterized. The quantitative real-time PCR and β-glucuronidase expression analysis revealed that CsSWEET5a is highly expressed in the anthers and pollen cells of male cucumber flowers from the microsporocyte stage (stage 9) to the mature pollen stage (stage 12). Its subcellular localization indicated that the CsSWEET5a protein is localized to the plasma membrane. The heterologous expression assays in yeast demonstrated that CsSWEET5a encodes a hexose transporter that can complement both glucose and fructose transport deficiencies. CsSWEET5a can significantly rescue the pollen viability and fertility of atsweet8 mutant Arabidopsis plants. The possible role of CsSWEET5a in supplying hexose to developing pollen cells via the apoplast is also discussed.

Changes in m6A RNA methylation are associated with male sterility in wolfberry

BackgroundN6-methyladenosine (m6A) modification is the most abundant type of RNA modification in eukaryotic cells, playing pivotal roles in multiple plant growth and development processes. Yet the potential role of m6A in conferring the trait of male sterility in plants remains unknown.ResultsIn this study, we performed RNA-sequencing (RNA-Seq) and m6A-sequencing (m6A-Seq) of RNAs obtained from the anther tissue of two wolfberry lines: ‘Ningqi No.1’ (LB1) and its natural male sterile mutant ‘Ningqi No.5’ (LB5). Based on the newly assembled transcriptome, we established transcriptome-wide m6A maps for LB1 and LB5 at the single nucleus pollen stage. We found that the gene XLOC_021201, a homolog of m6A eraser-related gene ALKBH10 in Arabidopsis thaliana, was significantly differentially expressed between LB1 and LB5. We also identified 1642 and 563 m6A-modified genes with hypermethylated and hypomethylated patterns, respectively, in LB1 compared with LB5. We found the hypermethylated genes significantly enriched in biological processes related to energy metabolism and lipid metabolism, while hypomethylation genes were mainly linked to cell cycle process, gametophyte development, and reproductive process. Among these 2205 differentially m6A methylated genes, 13.74% (303 of 2205) were differentially expressed in LB1 vis-à-vis LB5.ConclusionsThis study constructs the first m6A transcriptome map of wolfberry and establishes an association between m6A and the trait of male sterility in wolfberry.

RNA-seq analysis of synchronized developing pollen isolated from a single anther.

Pollen development, from unicellular microspores to anthesis, is a complex process involving the coordinated specification, differentiation and functions of different cell types. Key to understanding this development is identifying the genes expressed at precise stages of development. However, transcriptomic studies on pollen prior to anthesis are complicated by the inaccessible nature of pollen developing in the anther and the resistant pollen wall. To assist with understanding gene expression during pollen development we have developed a protocol to perform RNA-Seq on pollen isolated from a single anther (SA RNA-Seq). The protocol involves removing pollen from a single anther for analysis and viewing the remaining pollen to determine the developmental stage. The isolated pollen is chemically lysed and mRNA isolated from the lysate using an oligo-dT column before library preparation. Here, we report on the development and testing of our method and the generation of a transcriptome for three stages of pollen development from Arabidopsis (Arabidopsis thaliana) and two stages from male kiwifruit (Actinidia chinensis). This protocol enables the transcriptome of precise developmental stages of pollen to be analyzed, and uses a small number of plants, potentially facilitating studies that require a range of treatments or the analysis of the first generation of transgenic plants.

Ultrastructural changes in developmental stages of anther and pollen grains as affected by short-term exposure to low temperatures in strawberry

Short-term exposure to low temperatures can lower pollen viability and germination in strawberry. Fruit malformation was found to be negatively correlated with pollen germination. Ultrastructural analysis was performed at various developmental stages of anthers and pollen grains of the strawberry upon exposure to short-term low temperature (STLT). ‘Maehyang’ strawberry was exposed to 2 oC from 6 pm to 9 am for 3 days during different flower developments before anthesis, whereas the plants in control remained in the greenhouse. The epidermis did not develop well and was deformed after STLT. The epidermis showed a significantly lower cell expansion index in the short-term low-temperature treatment than in control at the vacuolated microspore, bicellular pollen, and mature pollen stages, indicating significant deformation of the epidermal cells by low temperature. The projected area of starch was significantly greater in STLT treatment than in control at the mature pollen stage. The relative fluorescence intensity of anthers was significantly higher in STLT treatment than in control at the sporogenous cell and microspore mother cell stages, indicating that low temperature substantially inhibited anther development at early stages. The results showed that STLT stress caused abnormal development of the ultrastructure of the anther wall and pollen grains, along with starch over-accumulation during pollen development. These abnormal developments may lead to poor pollen germination and thus cause fruit malformation in strawberry.

Developing regenerated rice lines by the anther culture method

When developing a variety, along with the classical breeding methods, there are increasingly being used bio-technological and molecular genetic methods. These methods include a cultivation technology of isolated anthers, based on the phenomenon of androgenesis. The current paper has presented the results of developing androgenetic rice lines from 7 hybrids of the first generation. The purpose of the work was to study the ability of rice hybrids to form callus on artificial nutrient media and develop regenerated plants followed by a visual estimation of ploidy. Developing dihaploid homozygous lines will speed up the breeding process. The selection of rice panicles was carried out in the field in the shooting phase at the stage of mononuclear pollen. There were cultivated 7218 anthers, of which there were obtained 259 calli. The combinations ‘Akustik x Nerica 1’, ‘Nerica 1 x Akustik c’, ‘Kapitan x Akustik’ were the most responsive to callus formation. The regeneration frequency to the number of calli was 9.13 %. There were identified 794 new formations, from which there were formed 46 plants, including 26 albino and 20 green. The largest number of plants was formed in the hybrid ‘Kapitan x Akustik’ (22 pcs., 14 of which are green). Among the survived green regenerated plants, according to a complex of morphological traits, two plants turned out to be haploids with small sterile flowers and anthers, thin and narrow leaves; five plants were dihaploids with well-grained panicles and medium-sized leaves; two plants were tetraploids that had a wide leaf, a large panicle with very large flowers, but with low fertility. Plants and seeds were handed over to breeders for further work.

Cytochemical and comparative transcriptome analyses elucidate the formation and ecological adaptation of three types of pollen coat in Zingiberaceae

BackgroundThe pollen ornate surface of flowering plants has long fascinated and puzzled evolutionary biologists for their variety. Each pollen grain is contained within a pollen wall consisting of intine and exine, over which the lipoid pollen coat lies. The cytology and molecular biology of the development of the intine and exine components of the pollen wall are relatively well characterised. However, little is known about the pollen coat, which confers species specificity. We demonstrate three types of pollen coat in Zingiberaceae, a mucilage-like pollen coat and a gum-like pollen coat, along with a pollen coat more typical of angiosperms. The morphological differences between the three types of pollen coat and the related molecular mechanisms of their formation were studied using an integrative approach of cytology, RNA-seq and positive selection analysis.ResultsContrary to the ‘typical’ pollen coat, in ginger species with a mucilage-like (Caulokaempferia coenobialis, Cco) or gum-like (Hornstedtia hainanensis, Hhn) pollen coat, anther locular fluid was still present at the bicellular pollen (BCP) stage of development. Nevertheless, there were marked differences between these species: there were much lower levels of anther locular fluid in Hhn at the BCP stage and it contained less polysaccharide, but more lipid, than the locular fluid of Cco. The set of specific highly-expressed (SHE) genes in Cco was enriched in the ‘polysaccharide metabolic process’ annotation term, while ‘fatty acid degradation’ and ‘metabolism of terpenoids and polyketides’ were significantly enriched in SHE-Hhn.ConclusionsOur cytological and comparative transcriptome analysis showed that different types of pollen coat depend on the residual amount and composition of anther locular fluid at the BCP stage. The genes involved in ‘polysaccharide metabolism’ and ‘transport’ in the development of a mucilage-like pollen coat and in ‘lipid metabolism’ and ‘transport’ in the development of a gum-like pollen coat probably evolved under positive selection in both cases. We suggest that the shift from a typical pollen coat to a gum-like or mucilage-like pollen coat in flowering plants is an adaptation to habitats with high humidity and scarcity of pollinators.

Identification of long non-coding RNAs and microRNAs involved in anther development in the tropical Camellia oleifera

BackgroundExplored the molecular science of anther development is important for improving productivity and overall yield of crops. Although the role of regulatory RNAs, including long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), in regulating anther development has been established, their identities and functions in Camellia oleifera, an important industrial crop, have yet not been clearly explored. Here, we report the identification and characterization of genes, lncRNAs and miRNAs during three stages of the tropical C. oleifera anther development by single-molecule real-time sequencing, RNA sequencing and small RNA sequencing, respectively.ResultsThese stages, viz. the pollen mother cells stage, tetrad stage and uninucleate pollen stage, were identified by analyzing paraffin sections of floral buds during rapid expansion periods. A total of 18,393 transcripts, 414 putative lncRNAs and 372 miRNAs were identified, of which 5,324 genes, 115 lncRNAs, and 44 miRNAs were differentially accumulated across three developmental stages. Of these, 44 and 92 genes were predicted be regulated by 37 and 30 differentially accumulated lncRNAs and miRNAs, respectively. Additionally, 42 differentially accumulated lncRNAs were predicted as targets of 27 miRNAs. Gene ontology enrichment indicated that potential target genes of lncRNAs were enriched in photosystem II, regulation of autophagy and carbohydrate phosphatase activity, which are essential for anther development. Functional annotation of genes targeted by miRNAs indicated that they are relevant to transcription and metabolic processes that play important roles in microspore development. An interaction network was built with 2 lncRNAs, 6 miRNAs and 10 mRNAs. Among these, miR396 and miR156 family were up-regulated, while their targets, genes (GROWTH REGULATING FACTORS and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE genes) and lncRNAs, were down-regulated. Further, the trans-regulated targets of these lncRNAs, like wall-associated kinase2 and phosphomannose isomerase1, are involved in pollen wall formation during anther development.ConclusionsThis study unravels lncRNAs, miRNAs and miRNA-lncRNA-mRNA networks involved in development of anthers of the tropical C. oleifera lays a theoretical foundation for further elucidation of regulatory roles of lncRNAs and miRNAs in anther development.

OsCPK29 interacts with MADS68 to regulate pollen development in rice

Pollen development and its germination are obligatory for the reproductive success of flowering plants. Calcium-dependent protein kinases (CPKs, also known as CDPKs) regulate diverse signaling pathways controlling plant growth and development. Here, we report the functional characterization of a novel OsCPK29 from rice, which is mainly expressed during pollen maturation stages of the anther. OsCPK29 exclusively localizes in the nucleus, and its N-terminal variable domain is responsible for retaining it in the nucleus. OsCPK29 knockdown rice plants exhibit reduced fertility, set fewer seeds, and produce collapsed non-viable pollen grains that do not germinate. Cytological analysis of anther semi-thin sections during different developmental stages suggested that pollen abnormalities appear after the vacuolated pollen stage. Detailed microscopic study of pollen grains further revealed that they were lacking the functional intine layer although exine layer was present. Consistent with that, downregulation of known intine development-related rice genes was also observed in OsCPK29 silenced anthers. Furthermore, it has been demonstrated that OsCPK29 interacts in vitro as well as in vivo with the MADS68 transcription factor which is a known regulator of pollen development. Therefore, phenotypic observations and molecular studies suggest that OsCPK29 is an important regulator of pollen development in rice.

Mitogen-activated protein kinase 4 is obligatory for late pollen and early fruit development in tomato.

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules regulating vegetative and reproductive development of plants. However, the molecular mechanisms of the SlMPK4 gene in tomato pollen and fruit development remain elusive. SlMPK4 is preferentially and highly expressed in tomato stamens and its mRNA levels increase during early flower development, peaking at the mature pollen stage. Either up- or downregulation of SlMPK4 expression had no significant effect on tomato vegetative growth. However, RNAi-mediated suppression of SlMPK4 caused defects in pollen development, resulting in pollen abortion. The aborted pollen grains were either malformed or collapsed and completely lacked viability, resulting in a predominantly reduced fruit set rate in RNAi lines compared with control and overexpressing transgenic plants. Interestingly, seed development was inhibited in RNAi lines. Moreover, >12% of emasculated RNAi flowers developed seedless fruits without pollination. Anthers can produce typical microspore mother cells as well as uninucleate microspores, according to cytological investigations, while binucleate pollen ceased to produce typical mature pollen. Pollen abortion was further confirmed by transmission electron microscopy analysis at the binucleate stage in RNAi plants. The exine layer in aberrant pollen had a normal structure, while the intine layer appeared thicker. Suppression of SlMPK4 affects the transcript level of genes related to cell wall formation and modification, cell signal transduction, and metabolic and biosynthetic processes. A subset of genes that may be putative substrates of plant MAPKs were also differentially changed in RNAi transgenic flowers. Taken together, these results suggest that SlMPK4 plays a critical role in regulating pollen development and fruit development in tomato plants.

DNA methylation of

In wheat (Triticum aestivum L.), S-type cytoplasmic male sterile (S-CMS) lines can mainly transform from sterility to fertility at the mononuclear pollen stage. Observations from microscopy revealed an inhibition of starch accumulation within the pollen grain, suggesting that an alteration in carbohydrate metabolism or assimilate supply may be involved in S-CMS pollen abortion. We measured levels of various carbohydrates and activities of key enzymes of sucrose metabolism at the mononuclear pollen stage in anthers collected from an S-CMS line and its maintainer line and found that nonreducing sugars increased in S-CMS anthers. Sucrose accounted for part of the nonreducing sugar accumulation. The activity of invertase declined significantly, whereas sucrose synthase activity during starch accumulation in pollen showed no significant change in S-CMS anthers at the mononuclear pollen stage. The results suggest that sucrose conversion and inhibition of invertase were probably responsible for the pollen abortion. Because there is a high correlation between the rate of ethylene evolution and sucrose content, ethylene anabolism was determined. TaACS2 regulates sucrose metabolism in pollen probably through catalysing the synthesis of ethylene precursor 1-aminocyclopropane-1-carboxylic acid in wheat. In this study, ethylene production of anthers underwent accumulation, revealed by gas chromatography, and expression levels of TaACS2 were upregulated in the S-CMS line, as determined by quantitative real-time PCR. We investigated the DNA methylation pattern of TaACS2 in the core promoter region using bisulfite genomic sequencing, and lower methylation was observed in the S-CMS line. These results suggest that DNA methylation of the TaACS2 gene may be involved in the sterility–fertility transition by regulating the synthesis of ethylene in S-CMS anthers.

Integrated analysis of small RNA, transcriptome and degradome sequencing reveals that micro-RNAs regulate anther development in CMS cotton

Cytoplasmic male sterility (CMS) is an important phenotypic trait for heterosis utilization in cotton (Gosspium hirsutum L.). Micro-RNAs (miRNAs) play crucial roles in the pollen development of plants. To elucidate the roles of miRNA-target gene pairs in the CMS cotton line ganmian15A and its maintainer line ganmian15B, an integrated small RNA (sRNA), transcriptome, and degradome sequencing analysis of these lines at the pollen abortion stage was performed in the present research. A total of 339 known miRNAs and 961 conserved miRNAs were identified through high-throughput sequencing, among which 124 were differentially expressed between ganmian15A and ganmian15B. By comprehensive analysis of miRNA, transcriptome, and degradome sequencing, we identified 11 pairs of miRNAs/target genes at the pollen abortion stage. Among them, 9 miRNAs (4 novel and 5 known) were found for the first time to be associated with the occurrence of male sterility in cotton. The target genes of miRna-38 and miRna-23 were linked to cell wall development at the pollen stage. They may play an imperative role in CMS cotton by delaying the degradation of tapetal cells. Our findings revealed new insights into the genetic control of CMS occurrence, which will support the breeding of new sterile lines of cotton in the future.

Interaction of gibberellin and other hormones in almond anthers: phenotypic and physiological changes and transcriptomic reprogramming

Low temperature causes anther dysfunction, severe pollen sterility and, ultimately, major yield losses in crop plants. Previous studies have shown that the gibberellic acid (GA) metabolic pathway plays an important role in this process by regulating tapetum function and pollen development. However, the interaction mechanism of GA with other hormones mediating anther development is still unclear. Herein, we collected and analyzed almond (Amygdalus communis L.) anthers at the meiosis, tetrad, 1-nucleus, and mature 2-nucleus stages. The growth rate per 1000 anthers exhibited a significant positive correlation with the total bioactive GA compound content, and the levels of all bioactive GA compounds were highest in the 1-nucleus pollen stage. GA3 treatment experiments indicated that exogenous GA3 increased the levels of indole-3-acetic acid (IAA), trans-zeatin (tZ), and jasmonic acid (JA) and decreased the levels of salicylic acid (SA) and abscisic acid (ABA); moreover, GA3 improved pollen viability and quantities under cold conditions, whereas PP333 (paclobutrazol, an inhibitor of GA biosynthesis) was antagonistic with GA3 in controlling anther development. RNA-seq and qRT-PCR results showed that GA played an important role in anther development by regulating the expression of other phytohormone pathway genes, dehydration-responsive element-binding/C-repeat binding factor (DREB1/CBF)-mediated signaling genes, and anther development pathway genes. Our results reveal the novel finding that GA interacts with other hormones to balance anther development under normal- and low-temperature conditions in almond.

Disruption of carbohydrate and proline metabolism in anthers under low temperature causes pollen sterility in chickpea

Chickpea (Cicer arietinum L.) is the third most important food legume globally and is an important source of high-quality proteins to both humans and animals. It is highly sensitive to low temperature (LT) resulting in large scale yield losses. LT induces flower abortion in chickpea by disruption of gamete development and induction of pollen sterility. However, molecular mechanisms governing LT induced pollen sterility in chickpea are unknown. Disruption of carbohydrate metabolism has been implicated in LT induced damage to crops whereas proline and antioxidants protect plants from LT stress. In this study, the impact of LT on carbohydrates, proline and enzymatic antioxidants and expression of genes of these metabolite pathways were investigated in anthers at vacuolated pollen stage. The study included two genotypes, a cold-sensitive (GPF2) and a cold-tolerant (ICC 16349) that differed for pollen sterility under LT. Metabolite profiling revealed a reduction in starch and proline content in GPF2 under LT, whereas these parameters did not change but non-reducing sugars increased in ICC 16349. At the transcriptional level, cold initially lowered the transcription of all genes (19) under study in GPF2 and 14 genes out of 19 in ICC 16349. Initial down-regulation was followed by differential reactivation of transcription machinery in GPF2 and ICC 16349 at 30 min. Under LT, expression of starch synthesis, starch degradation, sucrose synthesis and sucrose transport genes showed a contrasting response in the two genotypes, where GPF2 showed downregulation of these genes while ICC 16,349 showed upregulation. The proline transport gene (proline transporter 1) showed higher downregulation in GPF2 than ICC 16349. Overall, pollen viability under LT in ICC 16349 was linked to the maintenance of starch, reducing sugars and proline levels and over-expression of UDP glucose pyrophosphorylase and cell wall invertase, while these processes were compromised in the GPF2 possibly leading to pollen sterility.

Integrated Proteo-Transcriptomic Analyses Reveal Insights into Regulation of Pollen Development Stages and Dynamics of Cellular Response to Apple Fruit Crinkle Viroid (AFCVd)-Infection in Nicotiana tabacum.

Tobacco (Nicotiana tabacum) pollen is a well-suited model for studying many fundamental biological processes owing to its well-defined and distinct development stages. It is also one of the major agents involved in the transmission of infectious viroids, which is the primary mechanism of viroid pathogenicity in plants. However, some viroids are non-transmissible and may be possibly degraded or eliminated during the gradual process of pollen development maturation. The molecular details behind the response of developing pollen against the apple fruit crinkle viroid (AFCVd) infection and viroid eradication is largely unknown. In this study, we performed an integrative analysis of the transcriptome and proteome profiles to disentangle the molecular cascade of events governing the three pollen development stages: early bicellular pollen (stage 3, S3), late bicellular pollen (stage 5, S5), and 6 h-pollen tube (PT6). The integrated analysis delivered the molecular portraits of the developing pollen against AFCVd infection, including mechanistic insights into the viroid eradication during the last steps of pollen development. The isobaric tags for label-free relative quantification (iTRAQ) with digital gene expression (DGE) experiments led us to reliably identify subsets of 5321, 5286, and 6923 proteins and 64,033, 60,597, and 46,640 expressed genes in S3, S5, and PT6, respectively. In these subsets, 2234, 2108 proteins and 9207 and 14,065 mRNAs were differentially expressed in pairwise comparisons of three stages S5 vs. S3 and PT6 vs. S5 of control pollen in tobacco. Correlation analysis between the abundance of differentially expressed mRNAs (DEGs) and differentially expressed proteins (DEPs) in pairwise comparisons of three stages of pollen revealed numerous discordant changes in mRNA/protein pairs. Only a modest correlation was observed, indicative of divergent transcription, and its regulation and importance of post-transcriptional events in the determination of the fate of early and late pollen development in tobacco. The functional and enrichment analysis of correlated DEGs/DEPs revealed the activation in pathways involved in carbohydrate metabolism, amino acid metabolism, lipid metabolism, and cofactor as well as vitamin metabolism, which points to the importance of these metabolic pathways in pollen development. Furthermore, the detailed picture of AFCVd-infected correlated DEGs/DEPs was obtained in pairwise comparisons of three stages of infected pollen. The AFCVd infection caused the modulation of several genes involved in protein degradation, nuclear transport, phytohormone signaling, defense response, and phosphorylation. Intriguingly, we also identified several factors including, DNA-dependent RNA-polymerase, ribosomal protein, Argonaute (AGO) proteins, nucleotide binding proteins, and RNA exonucleases, which may plausibly involve in viroid stabilization and eradication during the last steps of pollen development. The present study provides essential insights into the transcriptional and translational dynamics of tobacco pollen, which further strengthens our understanding of plant-viroid interactions and support for future mechanistic studies directed at delineating the functional role of candidate factors involved in viroid elimination.

Ultrastructural characterization of microlipophagy induced by the interaction of vacuoles and lipid bodies around generative and sperm cells in Arabidopsis pollen

During pollen maturation, various organelles change their distribution and function during development as male gametophytes. We analyzed the behavior of lipid bodies and vacuoles involved in lipophagy in Arabidopsis pollen using serial section SEM and conventional TEM. At the bicellular pollen stage, lipid bodies in the vegetative cells lined up at the surface of the generative cell. Vacuoles then tightly attached, drew in, and degraded the lipid bodies and eventually occupied the space of the lipid bodies. Degradation of lipid began before transfer of the entire contents of the lipid body. At the tricellular stage, vacuoles instead of lipid bodies surrounded the sperm cells. The degradation of lipid bodies is morphologically considered microautophagy. The atg2-1 Arabidopsis mutant is deficient in one autophagy-related gene (ATG). In this mutant, the assembly of vacuoles around sperm cells was sparser than that in wild-type pollen. The deficiency of ATG2 likely prevents or slows lipid degradation, although it does not prevent contact between organelles. These results demonstrate the involvement of microlipophagy in the pollen development of Arabidopsis.

Effects of low temperature in spring on fertility of pollen and formation of grain number in wheat

<p indent=0mm>Grain yield and the number of grains per ear of wheat decreased under the low temperature after jointing stage. In order to explore the physiological reasons of the decrease of grains number per ear by low temperature in spring, the spring wheat variety Yangmai 16 and the semi-winter wheat variety Xumai 30 were used as tested varieties to analyze the effects of low temperature in spring on the fertility of pollen and the formation of grain numbers at the appearance of the penultimate leaf stage (5℃/–3℃, day/night), booting stage (8℃/–1℃, day/night), and anthesis stage (12℃/4℃, day/night). The results showed that low temperature at the appearance of the penultimate leaf stage and booting stage resulted in abnormal metabolism of starch and protein in the anther both at the binuclear and tri-nuclear pollen stages and delayed degradation of the tapetum. The low temperature at the booting stage caused abnormal meiosis of pollen mother cells, abnormalities in chromosome pairing, which affected the formation of male gametophyte. The abortion rate of pollen was more significantly increased under low temperature at the appearance of the penultimate leaf stage than that at the booting stage, and it was the minimum at anthesis stage. The abortion rate of pollen in Xumai 30 was higher than that in Yangmai 16. The low temperature at three stages had significant effects on the number of spikelets, the number of fertile spikelets and the number of grains per spikelet. The effect of low temperature at the appearance of the penultimate leaf stage on grains number per spikelet was the most significant. There was a significant positive correlation between the number of grains per spike and pollen fertility, which was affected by abnormal meiosis, delayed degradation of the tapetum, and undersupply of nutriment for anther development. Therefore, the decline of pollen fertility caused by low temperature in spring is the main reason for the decrease of grains number per spike.

Arabidopsis ECERIFERUM3 (CER3) Functions to Maintain Hydration for Pollen–Stigma Recognition During Fertilization

Plant stigmas distinguish the pollen grains and allow compatible ones to fertilize female gametes. To analyze the underlying mechanism, conditional male-sterile mutations with impaired pollen coat and pollen–stigma recognition were isolated. The mutant pollen could germinate in vitro but not in vivo, suggesting that they are viable. The mutant stigma cells that contacted their own pollen-generated callose, a carbohydrate typically produced in response to foreign pollen. The defects of pollen hydration and fertilization in the mutants were rescued by high humidity, indicating that hydration is required for pollen–stigma interaction. Pollination with mixture of wild type and mutant pollens demonstrated that the pollen–stigma recognition of mutant pollen was disrupted. The mutant plants exhibited lack of stem waxes and postgenital fusion between aerial floral organs. In addition, the mutant pollen was deficient in very-long-chain lipids and had excess tryphine. Transmission electron microscopy analysis showed that mutant pollen had almost the same surface structure as the wild type at bicellular pollen stage. However, abnormal plastoglobuli were observed in the plastids of the mutant tapetum, which was an indicative of altered lipid accumulation. CER3 transcripts were found in anther tapetum and microspores at development stage 9. Our data reveal that CER3 is required for biosynthesis of tryphine lipids which function to maintain hydration for pollen–stigma recognition during fertilization.

OsAPT1 a Pollen Preferentially Expressed Gene Is Essential for Pollen Tube Germination and Elongation in Rice

Pollen tube germination and elongation are important processes for double fertilization in flowering plants. However, little is known about the regulatory mechanism of pollen germination and tube growth in rice. Here, we reported that OsAPT1, a pollen-high expression gene and encoding a putative APT1 protein in Oryza sativa, regulating pollen tube germination and elongation in rice. Real-time quantitative PCR (qRT-PCR) and in situ hybridization analysis showed high expression level of OsAPT1 at late stage of microsporogenesis including late uninucleate stage, binucleate pollen stage, and mature pollen grains. OsAPT1 RNA interference (OsAPT1-RNAi) lines showed that the pollen tube germination and elongation rate were decreased. Compared with wild type (WT), the integrity and assembly of actin microfilaments were abnormal in hydrated pollen grains of OsAPT1-RNAi plants, and the fluorescence signals of actin microfilaments were altered in the pollen tube of OsAPT1-RNAi plants. GO analysis of the co-expressing genes of OsAPT1 showed that the GO terms such as pollination, cell growth, cellular cytoskeleton organization, and motor activity were enriched. In addition, the qRT-PCR results showed variations in the expression of some genes related to pollen tube growth. Overall, our results indicated that OsAPT1 is required for pollen tube germination and elongation in rice.

Transcriptome Analysis Reveals Important Transcription Factor Families and Reproductive Biological Processes of Flower Development in Celery (Apium graveolens L.)

There are few reports on the reproductive biology of celery, which produces small flowers in a long flowering period. Anther development was analyzed by paraffin sectioning and related genes were examined by transcriptome sequencing and qPCR. The development process was divided into nine stages based on the significant changes in the cell and tissue morphologies. These stages included: archesporial stage, sporogenous cell stage, microspore mother cell stage, dyad and tetrad stage, mononuclear microspore stage, late uninucleate microspore stage, binuclear cell stage, mature pollen stage, and dehiscence stage. A total of 1074 differentially expressed genes were identified by transcriptome sequencing in the early flower bud, middle flower bud, and early flowering period. Functional annotation indicated that these genes were involved in physiological and biochemical processes such as ribosomes metabolism, sugar metabolism, and amino acid metabolism. Transcription factors such as C2H2, AP2/ERF, bZIP, WRKY, and MYB played key regulatory roles in anther development and had different regulatory capabilities at various stages. The expression patterns based on qPCR and transcriptome data of the selected transcription factor genes showed consistency, suggesting that these genes played an important role in different flower development stages. These results provide a theoretical basis for molecular breeding of new celery varieties with pollen abortion. Furthermore, they have enriched research on the reproductive biology of celery and the Apiaceae family.