Pollen Cells Research Articles

Impact of green techniques on intricate cell wall structure of bee pollen to enhance functional characteristics and improve its in vitro digestibility.

Bee pollen is a nutrient-rich super food, but its rigid dual-layered structure limits nutrient release and absorption. The outer exine, composed of stress-resistant sporopollenin, and the inner intine, consisting of cellulose and pectin, form a barrier to digestive breakdown. This study investigates the potential of green techniques, specifically supercritical fluid extraction and ultrasonication, to disaggregate pollen cell walls, enhancing its bioavailability and maximizing nutrient utilization. Ultrasonication treated pollen (USTP) and supercritical fluid extraction-treated pollen (STP) demonstrated disruption, as evidenced by scanning electron microscopy imaging. In relation to scanning electron microscopy, techno-functional, antioxidant, and compositional analysis displayed a positive outcome, with crude lipid, protein, antioxidant activity (2,2-diphenyl-1-picrylhydrazyl activity and 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid assay) and total phenolic content increased by 34.80%, 32.58%, 10.80%, 11.37%, and 83.94%, respectively. Based on the above properties, USTP for 4 h and STP at 400 bar for 40 min were identified as the optimal conditions for disintegration. Furthermore, optimized samples analyzed for amino acid and mineral release revealed a notable increase in composition of essential amino acid and minerals (Ca, Cu, Fe, etc.) by ∼1.5 and 1.2 times, respectively. Along with significant changes in composition, fractured pollen exhibited 1.4 folds increase in protein digestibility with minor differences in thermal stability, and crystallinity as established by differential scanning calorimetry, and X-ray diffraction analysis. The study confirms that nutrient release and absorption remain restricted without pre-treatment, highlighting the necessity of specific treatment to disintegrate bee pollen before its use as a functional food ingredient. PRACTICAL APPLICATION: Bee pollen is a rich source of all the essential nutrients required by the humans and recognised as a complete food. However, its tough cellular structure restricts its utilisation in numerous food applications. Therefore, to disintegrate bee pollen and release its nutrients, ultrasonication and super critical fluid extraction processes were employed to improve its utilization for human purposes. Both the treatment techniques, enhanced bee pollen's bioavailability and functional properties, making it more suitable for use in nutraceuticals and functional foods.These treatments proved to increase the antioxidant capacity, digestibility, and create high-value ingredient for supplements, beverages, and fortified foods.

Morphological and Transcriptomic Analyses Reveal the Involvement of Key Metabolic Pathways in Male Sterility in Chimonanthus praecox (L.) Genotypes.

Chimonanthus praecox (Calycanthaceae family) is a unique ornamental and economic flowering tree in China, and after thousands of years of cultivation, it has produced several varieties and varietal types. Notably, male sterility is common in flowering plants and is an important tool for the genetic improvement in plants and optimization using hybrid plant technology; however, there have been no reports on male-sterile material or related studies on C. praecox. To our knowledge, this is the first time that C. praecox male sterility is dissected unveiling the involvement of key metabolic pathways. Notably, male sterility in C. praecox was observed during the budding period and likely occurred during the premature stage of pollen cell maturation. Additionally, differentially expressed genes in the starch and sucrose metabolism pathway and the plant hormone signal transduction pathway showed regular expression trends. This study reports on significant genetic differences that contribute to male sterility in C. praecox and provides a basis for further research and breeding strategies.

The MYC transcription factor PbrMYC8 negatively regulates PbrMSL5 expression to promote pollen germination in Pyrus

The successful germination of pollen is essential for double fertilization in flowering plants. Mechanosensitive channels of small conductance (MscS-like, MSL) inhibit pollen germination and maintains cellular integrity of pollen during this process. Therefore, it is vital to carefully regulate the expression of MSL to promote successful pollen germination. Despite its importance, the molecular mechanisms governing MSL expression in plants remain poorly understood. Here, we had identified 15 MSL genes in the pear, among which PbrMSL5 was expressed in pollen development. Subcellular localization experiments revealed that PbrMSL5 was located in both plasma membrane and cytoplasm. Functional investigations, including complementation experiments using the atmsl8 mutant background, demonstrated the involvement of PbrMSL5 in preserving pollen cell integrity and inhibiting germination. Antisense oligonucleotide experiments further confirmed that PbrMSL5 suppressed pear pollen germination by reducing osmotic pressure and Cl− content. Yeast one-hybrid, electrophoretic mobility shift assays, and dual luciferase reporter assay elucidated that PbrMYC8 interacts directly with the N-box element, leading to the suppression of PbrMSL5 expression and promoted pollen germination. These results represented a significant advancement in unraveling the molecular mechanisms controlling plant MSL expression. This study showed valuable contribution to advancing our comprehension of the mechanism underlying pollen germination.

Evaluating the efficiency of supplementary feeding as a management strategy for enhancing honeybee (Apis mellifera L.) colony growth and productivity

Sustaining honeybee colonies is challenging during dearth periods as their metabolic functions are reduced due to limited foraging activities. The experiment used honeybee colonies of Apis mellifera, and five different low-cost supplementary foods—sugar, banana, pumpkin, maize flour, and rice flour syrups—were introduced as treatments. Every box for each treatment received a daily 300-ml supplementary food syrup consisting of a specific amount of feeding materials along with 100 g of brown sugar and 20 g of honey. The amount of food consumed was assessed on the second day following the supplementation. Supplemental food with low-cost feeding materials significantly impacts the growth and strength of the colonies. Results revealed significant impacts on colony growth and strength, with all supplements contributing to food consumption over 78%. Despite variations in brood and pollen cells, all feeding supplements showcased efficiency in supporting honeybee feeding, indicating their potential utility in mitigating the challenges during the dearth period. Notably, pumpkin syrup emerged as the best supplement, offering cost-effectiveness compared to sugar and banana syrups, and it could reduce sugar syrup costs by 50% while enhancing brood, honey, and pollen cell production by 71.36%, 108.36%, and 58.73%, respectively. The findings of the economic analysis revealed that the cost of feeding materials was the highest for sugar syrup ($1.89), followed by banana ($0.91), pumpkin ($0.83), maize ($0.53), and rice ($0.53). This study suggests that supplementing honeybee colonies with low-cost feeding materials can positively impact colony growth and strength during dearth periods and advance the beekeeper’s decision as a cost-effective alternative to traditional sugar syrup.

Overriding Mendelian inheritance in Arabidopsis with a CRISPR toxin-antidote gene drive that impairs pollen germination.

Synthetic gene drives, inspired by natural selfish genetic elements and transmitted to progeny at super-Mendelian (>50%) frequencies, present transformative potential for disseminating traits that benefit humans throughout wild populations, even facing potential fitness costs. Here we constructed a gene drive system in plants called CRISPR-Assisted Inheritance utilizing NPG1 (CAIN), which uses a toxin-antidote mechanism in the male germline to override Mendelian inheritance. Specifically, a guide RNA-Cas9 cassette targets the essential No Pollen Germination 1 (NPG1) gene, serving as the toxin to block pollen germination. A recoded, CRISPR-resistant copy of NPG1 serves as the antidote, providing rescue only in pollen cells that carry the drive. To limit potential consequences of inadvertent release, we used self-pollinating Arabidopsis thaliana as a model. The drive demonstrated a robust 88-99% transmission rate over two successive generations, producing minimal resistance alleles that are unlikely to inhibit drive spread. Our study provides a strong basis for rapid genetic modification or suppression of outcrossing plant populations.

Heteroploid F1 hybrids generated from Coffea canephora × C. arabica interploidy crossing

Abstract Distant hybrids between two coffee species was considered promising for breeders to select novel genotypes highly resilient to changing global climate, despite pre- and or post-zygotic barriers. However, the different ploidy between C. arabica (2n=4x=44) and its diploid counterparts was another challenging barrier. The expected ploidy level for developed F1 hybrid was triploid. This study was aimed to identify ploidy level of F1 hybrids generated from Coffea canephora × C. arabica interploidy crossing. Ploidy determination was held using indirect methods, i.e. based on morphometric analysis of pollen and stomatal guard cells. Polar and equatorial diameter of 300 freshly collected pollen from each of six individuals of F1 hybrids were measured using ImageJ software. Stomatal frequency was measured as the number of guard cells on leaf area of 1 mm2. Pollen and stomatal guard cells of their diploid maternal and tetraploid paternal were also measured to be used as a standard for ploidy level determination. Generated datasets were immediately analysis statistically using t-Test procedure. The results shows that four of six F1 hybrid individuals had similar pollen size as well as stomatal density to diploid maternal. Meanwhile, another single individual was similar to tetraploid paternal. However, the rest of single individuals were similar to both of maternal and paternal. Based on those results, it could be inferred that there weres three different ploidy levels among F1 hybrid individuals, namely diploid, triploid, and tetraploid.

Pollen transcriptomic analysis provided insights into understanding the molecular mechanisms underlying grafting-induced improvement in potato fertility.

Heterologous grafting has been proven to be a valid approach to improving potato fertility, especially when grafting potatoes with other Solanaceae family plants. However, the mechanisms underlying grafting-induced improvement in potato fertility are still unknown. In this study, a poor-fertility potato cultivar "Qingshu No. 9" (Q9) was grafted with a tomato cultivar "Zhongyan988" (ZY988) to study the effects of heterologous grafting in the former. The tuber yield was controlled by different grafting and cultivation approaches, and the correlation between tuber yield and pollen vigor was studied. Comparative transcriptomic analysis of the potential mechanisms of pollen in potato scion fertility changes. Grafting with the tomato rootstock effectively promoted the flower and fruit formation in the scion potato and improved its pollen viability by 15%-20%. In addition, a significant negative correlation was observed between the potato tuber yield and pollen viability, suggesting a potential impact on the metabolic regulatory network related to tuber formation. From the comparative transcriptomic analysis between the pollens from Q9 self-grafted plants and Q9-tomato grafting scion, 513 differentially expressed genes (DEGs) were identified. These DEGs were found to be related to gametophyte and pollen development, carbohydrate metabolism, and protein processing. Thus, these DEGs might be involved in improved fertility after reduced tuberization in plants subjected to heterologous grafting. Potato/tomato heterologous grafting significantly improved the pollen viability of scion potatoes and was associated with the absence of potato tubers. Heterologous grafting promotes the transcription of genes related to protein processing, carbohydrate metabolism, and pollen development in pollen cells, resulting in the production of fertile pollen. Our results provided initial clues to understanding the improvement of potato fertility using the heterologous grafting method, which might be a useful tool in assisted potato breeding.

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.

MBD2 couples DNA methylation to transposable element silencing during male gametogenesis.

DNA methylation is an essential component of transposable element (TE) silencing, yet the mechanism by which methylation causes transcriptional repression remains poorly understood1-5. Here we study the Arabidopsis thaliana Methyl-CpG Binding Domain (MBD) proteins MBD1, MBD2 and MBD4 and show that MBD2 acts as a TE repressor during male gametogenesis. MBD2 bound chromatin regions containing high levels of CG methylation, and MBD2 was capable of silencing the FWA gene when tethered to its promoter. MBD2 loss caused activation at a small subset of TEs in the vegetative cell of mature pollen without affecting DNA methylation levels, demonstrating that MBD2-mediated silencing acts strictly downstream of DNA methylation. TE activation in mbd2 became more significant in the mbd5 mbd6 and adcp1 mutant backgrounds, suggesting that MBD2 acts redundantly with other silencing pathways to repress TEs. Overall, our study identifies MBD2 as a methyl reader that acts downstream of DNA methylation to silence TEs during male gametogenesis.

Identification and characterization of circular RNAs involved in the fertility stability of cotton CMS-D2 restorer line under heat stress

BackgroundAs a vital type of noncoding RNAs, circular RNAs (circRNAs) play important roles in plant growth and development and stress response. However, little is known about the biological roles of circRNAs in regulating the stability of male fertility restoration for cytoplasmic male sterility (CMS) conditioned by Gossypium harknessii cytoplasm (CMS-D2) cotton under high-temperature (HT) stress.ResultsIn this study, RNA-sequencing and bioinformatics analysis were performed on pollen grains of isonuclear alloplasmic near-isogenic restorer lines NH [N(Rf1rf1)] and SH [S(Rf1rf1)] with obvious differences in fertility stability under HT stress at two environments. A total of 967 circRNAs were identified, with 250 differentially expressed under HT stress. We confirmed the back-splicing sites of eight selected circRNAs using divergent primers and Sanger sequencing. Tissue-specific expression patterns of five differentially expressed circRNAs (DECs) were also verified by RT-PCR and qRT-PCR. Functional enrichment and metabolic pathway analysis revealed that the parental genes of DECs were significantly enriched in fertility-related biological processes such as pollen tube guidance and cell wall organization, as well as the Pentose and glucuronate interconversions, Steroid biosynthesis, and N-Glycan biosynthesis pathways. Moreover, we also constructed a putative circRNA-mediated competing endogenous RNA (ceRNA) network consisting of 21 DECs, eight predicted circRNA-binding miRNAs, and their corresponding 22 mRNA targets, especially the two ceRNA modules circRNA346-miR159a-MYB33 and circRNA484-miR319e-MYB33, which might play important biological roles in regulating pollen fertility stability of cotton CMS-D2 restorer line under HT stress.ConclusionsThrough systematic analysis of the abundance, characteristics and expression patterns of circRNAs, as well as the potential functions of their parent genes, our findings suggested that circRNAs and their mediated ceRNA networks acted vital biological roles in cotton pollen development, and might be also essential regulators for fertility stability of CMS-D2 restorer line under heat stress. This study will open a new door for further unlocking complex regulatory mechanisms underpinning the fertility restoration stability for CMS-D2 in cotton.

Remote focusing optical tweezers for 3D imaging.

We present a remote focusing optical tweezer utilizing a 4f symmetrical optical system to compensate the high-order aberration during annular light refocusing. The position of the optical trap can be adjusted beyond the range of one hundred micrometers in the axial direction by means of tuning the position of the mirror placed in the focal region of the illumination objective lens. This optical tweezer can be combined with a sectioning microscope to realize three-dimensional (3D) imaging, e.g., a confocal microscope using a single water immersion objective lens. All optical elements are placed in one side of the sample, which is very useful for application in fields such as radiation biology, where radiation or magnetism disturbance must be introduced on the other side of the sample. In the experiment, a 10 µm diameter silicon dioxide microsphere and pollen cells immersed in the water are translated along the axis using the optical tweezer and, meanwhile, the sectioning images are obtained using the confocal microscope.

Cytological and transcriptomic analyses provide insights into the pollen fertility of synthetic allodiploid Brassica juncea hybrids.

Imbalanced chromosomes and cell cycle arrest, along with down-regulated genes in DNA damage repair and sperm cell differentiation, caused pollen abortion in synthetic allodiploid Brassica juncea hybrids. Interspecific hybridization is considered to be a major pathway for species formation and evolution in angiosperms, but the occurrence of pollen abortion in the hybrids is common, prompting us to recheck male gamete development in allodiploid hybrids after the initial combination of different genomes. Here, we investigated the several key meiotic and mitotic events during pollen development using the newly synthesised allodiploid B. juncea hybrids (AB, 2n=2× =18) as a model system. Our results demonstrated the partial synapsis and pairing of non-homologous chromosomes concurrent with chaotic spindle assembly, affected chromosome assortment and distribution during meiosis, which finally caused difference in genetic constitution amongst the final tetrads. The mitotic cell cycle arrest during microspore development resulted in the production of anucleate pollen cells. Transcription analysis showed that sets of key genes regulating cyclin (CYCA1;2 and CYCA2;3), DNA damage repair (DMC1, NBS1 and MMD1), and ubiquitin-proteasome pathway (SINAT4 and UBC) were largely downregulated at the early pollen meiosis stages, and those genes involved in sperm cell differentiation (DUO1, PIRL1, PIRL9 and LBD27) and pollen wall synthesis (PME48, VGDH11 and COBL10) were mostly repressed at the late pollen mitosis stages in the synthetic allodiploid B. juncea hybrids (AB). In conclusion, this study elucidated the related mechanisms affecting pollen fertility during male gametophyte development at the cytological and transcriptomic levels in the synthetic allodiploid B. juncea hybrids.

Single Infrared Spectrum Enables Simultaneous Identification of (Bio)Chemical Nature and Particle Size of Microorganisms and Synthetic Microplastic Beads.

Populations of nearly identical chemical and biological microparticles include the synthetic microbeads used in cosmetic, biomedical, agri-food, and pharmaceutical industries as well as the class of living microorganisms such as yeast, pollen, and biological cells. Herein, we identify simultaneously the size and chemical nature of spherical microparticle populations with diameters larger than 1 μm. Our analysis relies on the extraction of both physical and chemical signatures from the same optical spectrum recorded using attenuated total reflection (ATR)-Fourier transform infrared (FTIR) spectroscopy. These signatures are the spectral resonances caused by the microparticles, which depend on their size and the absorption peaks revealing their chemical nature. We validate the method first on separated and mixed groups of spherical microplastic particles of two different diameters, where the method is used to calculate the diameter of the microspherical particles. Then, we apply the method to correctly identify and measure the diameter of Saccharomyces cerevisiae yeast cells. Theoretical simulations to help in understanding the effect of size distribution and dispersion support our results.

VAMP726 from maize and Arabidopsis confers pollen resistance to heat and UV radiation by influencing lignin content of sporopollenin

Sporopollenin in the pollen cell wall protects male gametophytes from stresses. Phenylpropanoid derivatives, including guaiacyl (G) lignin units, are known to be structural components of sporopollenin, but the exact composition of sporopollenin remains to be fully resolved. We analyzed the phenylpropanoid derivatives in sporopollenin from maize and Arabidopsis by thioacidolysis coupled with nuclear magnetic resonance (NMR) and gas chromatography–mass spectrometry (GC–MS). The NMR and GC–MS results confirmed the presence of p-hydroxyphenyl (H), G, and syringyl (S) lignin units in sporopollenin from maize and Arabidopsis. Strikingly, H units account for the majority of lignin monomers in sporopollenin from these species. We next performed a genome-wide association study to explore the genetic basis of maize sporopollenin composition and identified a vesicle-associated membrane protein (ZmVAMP726) that is strongly associated with lignin monomer composition of maize sporopollenin. Genetic manipulation of VAMP726 affected not only lignin monomer composition in sporopollenin but also pollen resistance to heat and UV radiation in maize and Arabidopsis, indicating that VAMP726 is functionally conserved in monocot and dicot plants. Our work provides new insight into the lignin monomers that serve as structural components of sporopollenin and characterizes VAMP726, which affects sporopollenin composition and stress resistance in pollen.

Establishment of Biotesting System to Study Features of Innovative Multifunctional Biotextile

Abstract An established biotesting system designed to discover specific features of innovative multifunctional biotextile, encompassing integrated silica dioxide and succinite (Baltic amber) particles, is founded on reproducible, reliable, and relatively fast methods. The main idea starting this study was to create a system of test methods devoted to identification of specific features of biotextile materials designed to preserve living organisms from adverse environmental factors like enhanced electromagnetic radiation of different frequencies, without use of vivarium animals. Cultures of the freshwater macrophyte duckweed (Lemna minor) line Sta2 and fruit flies (Drosophila melanogaster) were chosen as model systems suitable for the study of the influence of electromagnetic field (EMF) radiation. The experiments showed changes of phenotypic features and growth parameters of test objects, as well as induction of point mutations of DNA (for example, insertions or deletions in chloroplast DNA and nucleotide substitutions in nuclear genes). The responses of test organisms induced by EMF were studied using microscopy, flow cytometry, and DNA sequencing methods. On the cell level, a new fast flow cytometry method for biotextile testing was developed: immature gametic (pollen) cell cultures were used as a highly sensitive model system (plant gametic cell response is comparable to human neutrophil response) to study the influence of EMF radiation. The method was based on the measurement of differences of fluorescence intensity between group of cells experimentally affected by EMF radiation and non-affected cells.

Morphology, Morphometry, and Nest Structure of Tetragonula biroi (Hymenoptera: Meliponini) In Central Sulawesi

Stingless bees are social bees that live in colonies. Indonesia has a variety of stingless bee species that can be identified based on morphological, morphometric and nest structure characters. This study aims to describe the morphological, morphometric and nest structure characteristics of Stingless bee Tetragonula biroi from Sigi Regency. This study used the roaming method to find samples in nature, with a purposive sampling technique. Obtained morphological, morphometry and nest structure data were analyzed using the Principal Component Analysis (PCA) method with PAST4 software. The morphological characters of the worker bee Tetragonula biroi have a dominant black body, blackish brown abdomen, antennae with 11 flagellomeres, black thorax, black hair standing on the mesoscutum and mesoscutellum, brownish black mandibles, with 5 hamuli. The most dominant character in the formation of seven groups of stingless bees in this study, namely Hamuli Number (HN), Fore Wing Length (FWL), and Length of Forewing Including Tegula (WL1). The nest entrance hole of T. biroi is elliptical in shape with a width of 7.1 cm and a height of 2.8 cm and is light brown in color. The internal structure of the nest of T. biroi consists of honey cells, pollen cells and stem cells. The existence of size differences in the same specimen and from different places, can be influenced by several environmental factors and is also a form of morphological adaptation.

The hydroxyproline O-arabinosyltransferase FIN4 is required for tomato pollen intine development.

The pollen grain cell wall is a highly specialized structure composed of distinct layers formed through complex developmental pathways. The production of the innermost intine layer, composed of cellulose, pectin and other polymers, is particularly poorly understood. Here we demonstrate an important and specific role for the hydroxyproline O-arabinosyltransferase (HPAT) FIN4 in tomato intine development. HPATs are plant-specific enzymes which initiate glycosylation of certain cell wall structural proteins and signaling peptides. FIN4 was expressed throughout pollen development in both the developing pollen and surrounding tapetal cells. A fin4 mutant with a partial deletion of the catalytic domain displayed significantly reduced male fertility in vivo and compromised pollen hydration and germination in vitro. However, fin4 pollen that successfully germinated formed morphologically normal pollen tubes with the same growth rate as the wild-type pollen. When we examined mature fin4 pollen, we found they were cytologically normal, and formed morphologically normal exine, but produced significantly thinner intine. During intine deposition at the late stages of pollen development we found fin4 pollen had altered polymer deposition, including reduced cellulose and increased detection of pectin, specifically homogalacturonan with both low and high degrees of methylesterification. Therefore, FIN4 plays an important role in intine formation and, in turn pollen hydration and germination and the process of intine formation involves dynamic changes in the developing pollen cell wall.

Ultrastructural study of anther parasitism of Ficus laevigata by Ficophagus laevigatus (Aphelenchoididae).

Transmission electron microscopy (TEM) was used to compare the ultrastructural differences between healthy male florets (anthers) and one floret parasitized by Ficophagus laevigatus in late phase C syconia of Ficus laevigata from southern Florida. Previous light-microscopic examination of paraffin-sectioned material showed that F. laevigatus-infested anthers of F. laevigata manifested as malformed, often with aberrant pollen and hypertrophied epidermal cells closest to regions containing propagating nematodes. Female florets or fig wasp-parasitized female florets were not observed to be parasitized by nematodes. Considering that plant-feeding in the Aphelenchoididae is purportedly much less specialized than in certain groups of the Tylenchomorpha, where specialized hypertrophied feeder cells are produced in response to nematode feeding, we examined the putative induced response in this unusual aphelenchoidid system with the higher resolution afforded by TEM. TEM confirmed the expression of significant epidermal cell hypertrophy of the anther and anther filament in the presence of propagating nematodes, which was expressed as cell enlargement (2-5X), fractionation of large electron-dense stores into smaller aggregates, irregularly shaped nuclei enclosed by an elongated nuclear envelope, nucleolus enlargement, increased organelle production, and apparent metabolism with increased numbers of mitochondria, pro-plastids, and endoplasmic reticulum, as well as increased thickening of the cell walls. Pathological effects were observed in adjacent cells/tissue (e.g., anther and anther filament parenchymal cells, pollen tubes, pollen, and endothecium) with apparent diminishment as the distance from propagating nematodes increased (which was also probably affected by number of nematodes). Some TEM sections captured previously undocumented ultrastructural highlights of propagating individuals of F. laevigatus.

The full-length Auxin Response Factor 8 isoform ARF8.1 controls pollen cell wall formation and directly regulates TDF1, AMS and MS188 expression.

Auxin Response Factor 8 plays a key role in late stamen development: its splice variants ARF8.4 and ARF8.2 control stamen elongation and anther dehiscence. Here, we characterized the role of ARF8 isoforms in pollen fertility. By phenotypic and ultrastructural analysis of arf8-7 mutant stamens, we found defects in pollen germination and viability caused by alterations in exine structure and pollen coat deposition. Furthermore, tapetum degeneration, a prerequisite for proper pollen wall formation, is delayed in arf8-7 anthers. In agreement, the genes encoding the transcription factors TDF1, AMS, MS188 and MS1, required for exine and pollen coat formation, and tapetum development, are downregulated in arf8-7 stamens. Consistently, the sporopollenin content is decreased, and the expression of sporopollenin synthesis/transport and pollen coat protein biosynthetic genes, regulated by AMS and MS188, is reduced. Inducible expression of the full-length isoform ARF8.1 in arf8-7 inflorescences complements the pollen (and tapetum) phenotype and restores the expression of the above transcription factors. Chromatin immunoprecipitation-quantitative polymerase chain reaction assay revealed that ARF8.1 directly targets the promoters of TDF1, AMS and MS188. In conclusion, the ARF8.1 isoform controls pollen and tapetum development acting directly on the expression of TDF1, AMS and MS188, which belong to the pollen/tapetum genetic pathway.

ZmMS39 encodes a callose synthase essential for male fertility in maize (Zea mays L.)

Callose contributes to many biological processes of higher plants including pollen development, cell plate and vascular tissue formation, as well as regulating the transport function of plasmodesmata. The functions of callose synthase genes in maize have been little studied. We describe a maize male-sterile mutant 39 (ms39) characterized by reduced plant height. In this study, we confirmed using CRISPR/Cas9 technology that a mutation in Zm00001d043909 (ZmCals12), encoding a callose synthase, is responsible for the male sterility of the ms39 mutant. Compared with male-fertile plants, callose deposition around the dyads and tetrads in ms39 anthers was significantly reduced. Increased cell autophagy observed in ms39 anthers may have been due to the premature programmed cell death of tapetal cells, leading to collapse of the anther wall structure. Disordered glucose metabolism in ms39 may have intensified autophagy in anthers. Evaluation of the ms39 gene on maize heterosis by paired-crossed experiment with 11 maize inbred lines indicated that ms39 can be used for maize hybrid seed production.