Three-dimensional label-free imaging and analysis of Pinus pollen grains using optical diffraction tomography.
ABSTRACT: The structure of pollen grains is related to the reproductive function of the plants. Here, three-dimensional (3D) refractive index maps were obtained for individual conifer pollen grains using optical diffraction tomography (ODT). The 3D morphological features of pollen grains from pine trees were investigated using measured refractive index maps, in which distinct substructures were clearly distinguished and analyzed. Morphological and physiochemical parameters of the pollen grains were quantified from the obtained refractive index (RI) maps and used to quantitatively study the interspecific differences of pollen grains from different strains. Our results demonstrate that ODT can assess the structure of pollen grains. This label-free and rapid 3D imaging approach may provide a new platform for understanding the physiology of pollen grains.
Project description:We present a multimodal approach for measuring the three-dimensional (3D) refractive index (RI) and fluorescence distributions of live cells by combining optical diffraction tomography (ODT) and 3D structured illumination microscopy (SIM). A digital micromirror device is utilized to generate structured illumination patterns for both ODT and SIM, which enables fast and stable measurements. To verify its feasibility and applicability, the proposed method is used to measure the 3D RI distribution and 3D fluorescence image of various samples, including a cluster of fluorescent beads, and the time-lapse 3D RI dynamics of fluorescent beads inside a HeLa cell, from which the trajectory of the beads in the HeLa cell is analyzed using spatiotemporal correlations.
Project description:<h4>Background and aims</h4>Understanding the species composition of pollen on pollinators has applications in agriculture, conservation and evolutionary biology. Current identification methods, including morphological analysis, cannot always discriminate taxa at the species level. Recent advances in flow cytometry techniques for pollen grains allow rapid testing of large numbers of pollen grains for DNA content, potentially providing improved species resolution.<h4>Methods</h4>A test was made as to whether pollen loads from single bees (honey-bees and bumble-bees) could be classified into types based on DNA content, and whether good estimates of proportions of different types could be made. An examination was also made of how readily DNA content can be used to identify specific pollen species.<h4>Key results</h4>The method allowed DNA contents to be quickly found for between 250 and 9391 pollen grains (750-28 173 nuclei) from individual honey-bees and between 81 and 11 512 pollen grains (243-34 537 nuclei) for bumble-bees. It was possible to identify a minimum number of pollen species on each bee and to assign proportions of each pollen type (based on DNA content) present.<h4>Conclusions</h4>The information provided by this technique is promising but is affected by the complexity of the pollination environment (i.e. number of flowering species present and extent of overlap in DNA content). Nevertheless, it provides a new tool for examining pollinator behaviour and between-species or cytotype pollen transfer, particularly when used in combination with other morphological, chemical or genetic techniques.
Project description:Upon release from the anther, pollen grains of angiosperm flowers are exposed to a dry environment and dehydrate. To survive this process, pollen grains possess a variety of physiological and structural adaptations. Perhaps the most striking of these adaptations is the ability of the pollen wall to fold onto itself to prevent further desiccation. Roger P. Wodehouse coined the term harmomegathy for this folding process in recognition of the critical role it plays in the survival of the pollen grain. There is still, however, no quantitative theory that explains how the structure of the pollen wall contributes to harmomegathy. Here we demonstrate that simple geometrical and mechanical principles explain how wall structure guides pollen grains toward distinct folding pathways. We found that the presence of axially elongated apertures of high compliance is critical for achieving a predictable and reversible folding pattern. Moreover, the intricate sculpturing of the wall assists pollen closure by preventing mirror buckling of the surface. These results constitute quantitative structure-function relationships for pollen harmomegathy and provide a framework to elucidate the functional significance of the very diverse pollen morphologies observed in angiosperms.
Project description:This review summarizes the available data related to the effects of air pollution on pollen grains from different plant species. Several studies carried out either on in situ harvested pollen or on pollen exposed in different places more or less polluted are presented and discussed. The different experimental procedures used to monitor the impact of pollution on pollen grains and on various produced external or internal subparticles are listed. Physicochemical and biological effects of artificial pollution (gaseous and particulate) on pollen from different plants, in different laboratory conditions, are considered. The effects of polluted pollen grains, subparticles, and derived aeroallergens in animal models, in in vitro cell culture, on healthy human and allergic patients are described. Combined effects of atmospheric pollutants and pollen grains-derived biological material on allergic population are specifically discussed. Within the notion of "polluen," some methodological biases are underlined and research tracks in this field are proposed.
Project description:We examined pollen grains and starch granules from a large number of modern populations of teosinte (wild Zea spp.), maize (Zea mays L.), and closely related grasses in the genus Tripsacum to assess their strengths and weaknesses in studying the origins and early dispersals of maize in its Mesoamerican cradle of origin. We report new diagnostic criteria and question the accuracy of others used previously by investigators to identify ancient maize where its wild ancestor, teosinte, is native. Pollen grains from teosinte overlap in size with those of maize to a much greater degree than previously reported, making the differentiation of wild and domesticated maize in palynological studies difficult. There is presently no valid method for separating maize and teosinte pollen on a morphological basis. Starch grain analysis, a recently developed tool of archaeobotany, appears to be of significant utility in distinguishing the seeds of teosinte from maize. We propose that the differences in starch grain morphology and size between wild and domesticated maize defined in this study may be associated with domestication genes in Zea that have been documented in the starch biosynthesis pathway. As previously reported, phytoliths effectively discriminate the female reproductive structures of Tripsacum, teosinte, and maize. Multiproxy microfossil studies of archaeological and paleoecological contexts appear to be effective tools for investigating the earliest stages of maize domestication and dispersals.
Project description:Pollen germination, along with pollen tube growth, is an essential process for the reproduction of flowering plants. The germinating pollen with tip-growth characteristics provides an ideal model system for the study of cell growth and morphogenesis. As an essential step towards a detailed understanding of this important process, the objective of this study was to comprehensively analyze the transcriptome changes during pollen germination and pollen tube growth. Using Affymetrix Arabidopsis ATH1 Genome Arrays, this study is the first to show the changes in the transcriptome from desiccated mature pollen grains to hydrated pollen grains and then to pollen tubes of Arabidopsis thaliana. The number of expressed genes, either for total expressed genes or for specifically expressed genes, increased significantly from desiccated mature pollen to hydrated pollen and again to growing pollen tubes, which is consistent with the finding that pollen germination and tube growth was significantly inhibited in vitro by a transcriptional inhibitor. The results of GO analyses showed that expression of genes related to cell rescue, transcription, signal transduction and cellular transport were significantly changed, especially for up-regulation, during pollen germination and tube growth, respectively. In particular, genes of the CaM/CML, CHX and Hsp families showed the most significant changes during pollen germination and tube growth. These results demonstrate that the overall transcription of genes, both in the number of expressed genes and in the levels of transcription, was increased. Furthermore, the appearance of many novel transcripts during pollen germination as well as tube growth indicates that these newly expressed genes may function in this complex process. SUBMITTER_CITATION: Yi Wang, Wen-Zheng Zhang, Lian-Fen Song, Jun-Jie Zou, Zhen Su, and Wei-Hua Wu. Transcriptome analyses show changes in gene expression to accompany pollen germination and tube growth in Arabidopsis. Plant Physiol. September 5, 2008; 10.1104/pp.108.126375 Experiment Overall Design: Three samples are analyzed in this experiment. They are desiccated mature pollen grains (MP), hydrated pollen grains (HP) and growing pollen tubes (PT) of Arabidopsis thaliana, respectively. Each sample has two biological replicates, so that there are 6 data sets of ATH1 array in this experiment.
Project description:Global temperature increase poses a serious challenge for agricultural production worldwide, affecting yield in many crops including vegetable crops. While most crop plants can survive temperature increases during their vegetative growth periods, the reproduction phase is highly heat-stress (HS)-sensitive. Impaired pollen development and functioning under HS is implicated as the major cause for yield reduction. To better understand HS effect on pollen and identify pollen thermotolerance mechanisms, we established conditions that enable developing pollen grains to acquire thermotolerance (ATT conditions), using tomato as a model system. High-throughput sequencing at cDNA level was performed by Massive Analysis of 3’cDNA using Illumina HiSeq 2000 technology, generating a total of 6430 and 4660 transcripts differentially expressed (p ≤ 1e-05) during pollen development/maturation and following response of developing pollen to ATT, respectively. Gene Onthology functional analysis showed that transcripts related to maintenance of protein homeostasis (translation, proteolysis, protein folding) were enriched during pollen maturation and following the ATT treatment in our study, highlighting these processes as central for enabling pollen maturation and maintenance of pollen functioning under HS. The transcriptomic data was compared to available pollen proteomic data based on the same experimental setup and an overlap of 47% was detected between differentially expressed proteins and transcripts following ATT conditions, highlighting genes/proteins involved in protein folding, oxidation-reduction and translation, and validating transcriptomic results. Involvement of mitochondria and endoplasmic reticulum in pollen heat acclimation, and activation of several HSPs including sHSPs and HSP101, for protecting pollen cellular components including the translational machinery, are indicated. The results of this study can serve as a valuable resource of genes for future research on improving pollen thermotolerance. Overall design: Establishment of the capacity of developing tomato pollen grains to acquire thermotolerance (ATT) using greenhouse-grown plants. Then, use of the established ATT conditions to test the effect on expression profiles of developing/maturing and mature pollen grains. Developing pollen grains are represented by pooling three developmental stages (3, 2 and 1 days before flower opening).
Project description:The pollination mechanism of most genera of the Podocarpaceae involves inverted ovules, a pollination drop and bisaccate pollen grains. Saccate grains have sometimes been referred to as 'non-wettable' due to their buoyant properties, while non-saccate pollen grains have been described as 'wettable'. The hydrodynamic properties of saccate pollen grains of seven podocarp species in five genera, Dacrydium Sol. ex G. Forst., Dacrycarpus (Endl.) de Laub., Manoao Molloy, Podocarpus L'Hér. ex Pers. and Prumnopitys Phil. have been tested in water, together with saccate and non-saccate pollen of four other conifer genera, Cedrus Trew (Pinaceae), Cephalotaxus Siebold & Zucc. ex Endl. (Cephalotaxaceae), Cupressus L. (Cupressaceae) and Phyllocladus Rich. ex Mirb. (Phyllocladaceae), and spores of three fern species and one lycopod species. All four spore types studied were non-wettable, whereas the bisaccate and trisaccate pollen types, like all other conifer pollen types, were wettable, enabling the grains to cross the surface tension barrier of water. Once past this barrier, grain behaviour was governed by presence or absence of sacci. Non-saccate and vestigially saccate grains sank, whereas saccate grains behaved like air bubbles, floating up to the highest point. In addition, the grains were observed to float in water with sacci uppermost, consistent with the suggestion that distally placed sacci serve to orientate the germinal furrow of the pollen grain towards the nucellus of an inverted ovule. Observations of pollen grains in the pollen chambers of naturally pollinated Prumnopitvs ovules confirmed this. The combination of buoyancy and wettability in saccate pollen has implications for the efficiency of the typical podocarp pollination mechanism.
Project description:Cadmium (Cd) is highly toxic, even at very low concentrations, to both animals and plants. Pollen is extremely sensitive to heavy metal pollutants; however, less attention has been paid to the protection of this vital part under heavy metal stress. A pot experiment was designed to investigate the effect of foliar application of Se (1 mg/L) and Mo (0.3 mg/L) either alone or in combination on their absorption, translocation, and their impact on Cd uptake and its further distribution in Brassica napus, as well as the impact of these fertilizers on the pollen grains morphology, viability, and germination rate in B. napus under Cd stress. Foliar application of either Se or Mo could counteract Cd toxicity and increase the plant biomass, while combined application of Se and Mo solutions on B. napus has no significant promotional effect on plant root and stem, but reduces the seeds' weight by 10?11%. Se and Mo have decreased the accumulated Cd in seeds by 6.8% and 9.7%, respectively. Microscopic studies, SEM, and pollen viability tests demonstrated that pollen grains could be negatively affected by Cd, thus disturbing the plant fertility. Se and Mo foliar application could reduce the toxic symptoms in pollen grains when the one or the other was sprayed alone on plants. In an in vitro pollen germination test, 500 ?M Cd stress could strongly inhibit the pollen germination rate to less than 2.5%, however, when Se (10 ?M) or Mo (1.0 ?M) was added to the germination medium, the rate increased, reaching 66.2% and 39.4%, respectively. At the molecular level, Se and Mo could greatly affect the expression levels of some genes related to Cd uptake by roots (IRT1), Cd transport (HMA2 and HMA4), Cd sequestration in plant vacuoles (HMA3), and the final Cd distribution in plant tissue at the physiological level (PCS1).
Project description:Seed yield, a major determinant for the commercial success of grain crops, critically depends on pollen viability, which is dramatically reduced by environmental stresses, such as drought, salinity, and extreme temperatures. Salinity, in particular, is a major problem for crop yield known to affect about 20% of all arable land and cause huge economic losses worldwide. Flowering plants are particularly sensitive to environmental stress during sexual reproduction, and even a short exposure to stressing conditions can severely hamper reproductive success, and thus reduce crop yield. Since proline is required for pollen fertility and accumulates in plant tissues in response to different abiotic stresses, a role of proline in pollen protection under salt stress conditions can be envisaged. In this perspective, we analyze old and new data to evaluate the importance of pollen development under saline conditions, and discuss the possibility of raising proline levels in pollen grains as a biotechnological strategy to stabilize seed yield in the presence of salt stress. The overall data confirm that proline is necessary to preserve pollen fertility and limit seed loss under stressful conditions. However, at present, we have not enough data to conclude whether or not raising proline over wildtype levels in pollen grains can effectively ameliorate seed yield under saline conditions, and further work is still required.