白皮松（Pinus bungeana）和青杄（Picea wilsonii）花粉脱壁技术建立及细胞学研究

花粉是种子植物受精过程中雄性生殖细胞的载体，在有性生殖过程中起着非常重要的作用。花粉与柱头、花柱、子房等都有相互作用，起识别作用的是细胞壁蛋白，而花粉粒的壁有明显的两层：内壁和外壁。裸子植物花粉与被子植物相比具有萌发时间长、生长缓慢、授粉－受精时间长等特点。花粉的内壁与外壁在花粉萌发和生长过程中的功能仍然缺乏研究，但是迄今为止现有的分子生物学技术在获得裸子植物花粉壁突变体方面并没有发挥作用。本研究以裸子植物白皮松(Pinus bungeana)和青杄（Picea wilsonii）花粉为材料建立裸子植物花粉脱壁的体系，包 括脱外壁花粉和花粉原生质体的制备，即将花粉的两层壁逐层剥离，并在此基础上进行了细胞学研究。 本文首先建立了裸子植物脱外壁花粉的分离技术。实验表明低浓度的酶组合短暂酶解后，辅以机械研磨，可以释放出大量的脱外壁花粉。其中在这两种裸子植物中的处理稍有不同，白皮松的脱外壁花粉在含12％蔗糖，0.5%纤维素酶和0.3%离析酶的溶液，pH 5.8 ，孵育半小时，辅以一定力度机械研磨的方法获得，而青杄脱外壁花粉的条件是0.8%纤维素酶和0.5%离析酶、12％蔗糖的溶液，其它条件类似。 其次我们成功建立了一种快速、有效、可重复的分离白皮松和青杄花粉原生质体的系统，分离频率可高达70%。对白皮松而言，最好的分离条件是2% 纤维素酶、1.5%离析酶、15%蔗糖、pH 5.8，黑暗、24℃条件下轻微振荡、酶解6 小时。3％纤维素酶和2％离析酶组合比较适宜青杄花粉原生质体的分离，同时需要较低浓度的蔗糖溶液（12％）。青杄花粉原生质体要远大于白皮松花粉原生质体，前者直径为80µm，后者为40µm。强烈的FDA 荧光显示很好的活性。在制备的过程中，酶的浓度、酶的配比、酶解时间、渗透压调节剂、起始材料对分离率都有影响。 运用免疫荧光标记技术显示，脱外壁花粉的表面存在纤维素、果胶质、阿拉伯半乳聚糖蛋白（AGPs）和凝集素结合位点，其中纤维素在整个表面都有分布，但在萌发孔附近的荧光最强;白皮松脱外壁花粉表面有胼胝质的存在，主要位于靠近气囊的部位，而青杄脱外壁花粉表面未能检测到胼胝质;酸性果胶质在白皮松脱外壁花粉靠近萌发孔处的荧光稍弱于其它部位，而在青杄脱外壁花粉的表面近极端的荧光要强于远极端;白皮松脱外壁花粉表面有酯化果胶的沉积，而青杄脱外壁花粉表面缺失酯化果胶;白皮松和青杄两种植物脱外壁花粉表面均有阿拉伯半乳糖的分布，而白皮松脱外壁花粉的荧光要远强于青杄;白皮松和青杄脱外壁花粉表面有伴刀豆凝集素（Conconavalin agglutinin, Con A ）和大豆凝集素（soybean agglutinin, SBA ）结合位点的分布，而缺失麦胚凝集素（wheat germ agglutinin, WGA）结合位点。另外，傅立叶红外光谱(FTIR) 分析结果也同样支持上述结论。

Cell wall composition regulates cell shape and growth behaviour in pollen tubes

L’une des particularités fondamentales caractérisant les cellules végétales des cellules animales est la présence de la paroi cellulaire entourant le protoplaste. La paroi cellulaire joue un rôle primordial dans (1) la protection du protoplaste, (2) est impliquée dans les mécanismes de filtration et (3) est le lieu de maintes réactions biochimiques nécessaires à la régulation du métabolisme et des propriétés mécaniques de la cellule. Les propriétés locales d’élasticité, d’extensibilité, de plasticité et de dureté des composants pariétaux déterminent la géométrie et la forme des cellules lors des processus de différentiation et de morphogenèse. Le but de ma thèse est de comprendre les rôles que jouent les différents composants pariétaux dans le modelage de la géométrie et le contrôle de la croissance des cellules végétales. Pour atteindre cet objectif, le modèle cellulaire sur lequel je me suis basé est le tube pollinique ou gamétophyte mâle. Le tube pollinique est une protubérance cellulaire qui se forme à partir du grain de pollen à la suite de son contact avec le stigmate. Sa fonction est la livraison des cellules spermatiques à l’ovaire pour effectuer la double fécondation. Le tube pollinique est une cellule à croissance apicale, caractérisée par la simple composition de sa paroi et par sa vitesse de croissance qui est la plus rapide du règne végétal. Ces propriétés uniques font du tube pollinique le modèle idéal pour l’étude des effets à courts termes du stress sur la croissance et le métabolisme cellulaire ainsi que sur les propriétés mécaniques de la paroi. La paroi du tube pollinique est composée de trois composantes polysaccharidiques : pectines, cellulose et callose et d’une multitude de protéines. Pour comprendre les effets que jouent ces différents composants dans la régulation de la croissance du tube pollinique, j’ai étudié les effets de mutations, de traitements enzymatiques, de l’hyper-gravité et de la gravité omni-directionnelle sur la paroi du tube pollinique. En utilisant des méthodes de modélisation mathématiques combinées à de la biologie moléculaire et de la microscopie à fluorescence et électronique à haute résolution, j’ai montré que (1) la régulation de la chimie des pectines est primordiale pour le contrôle du taux de croissance et de la forme du tube et que (2) la cellulose détermine le diamètre du tube pollinique en partie sub-apicale. De plus, j’ai examiné le rôle d’un groupe d’enzymes digestives de pectines exprimées durant le développement du tube pollinique : les pectate lyases. J’ai montré que ces enzymes sont requises lors de l’initiation de la germination du pollen. J’ai notamment directement prouvé que les pectate lyases sont sécrétées par le tube pollinique dans le but de faciliter sa pénétration au travers du style.

Changes to airborne pollen counts across Europe

A progressive global increase in the burden of allergic diseases has affected the industrialized world over the last half century and has been reported in the literature. The clinical evidence reveals a general increase in both incidence and prevalence of respiratory diseases, such as allergic rhinitis (common hay fever) and asthma. Such phenomena may be related not only to air pollution and changes in lifestyle, but also to an actual increase in airborne quantities of allergenic pollen. Experimental enhancements of carbon dioxide (CO) have demonstrated changes in pollen amount and allergenicity, but this has rarely been shown in the wider environment. The present analysis of a continental-scale pollen data set reveals an increasing trend in the yearly amount of airborne pollen for many taxa in Europe, which is more pronounced in urban than semi-rural/rural areas. Climate change may contribute to these changes, however increased temperatures do not appear to be a major influencing factor. Instead, we suggest the anthropogenic rise of atmospheric CO levels may be influential.

Pollen image classification using the classifynder system: Algorithm comparison and a case study on New Zealand honey

We describe an investigation into how Massey University’s Pollen Classifynder can accelerate the understanding of pollen and its role in nature. The Classifynder is an imaging microscopy system that can locate, image and classify slide based pollen samples. Given the laboriousness of purely manual image acquisition and identification it is vital to exploit assistive technologies like the Classifynder to enable acquisition and analysis of pollen samples. It is also vital that we understand the strengths and limitations of automated systems so that they can be used (and improved) to compliment the strengths and weaknesses of human analysts to the greatest extent possible. This article reviews some of our experiences with the Classifynder system and our exploration of alternative classifier models to enhance both accuracy and interpretability. Our experiments in the pollen analysis problem domain have been based on samples from the Australian National University’s pollen reference collection (2,890 grains, 15 species) and images bundled with the Classifynder system (400 grains, 4 species). These samples have been represented using the Classifynder image feature set.We additionally work through a real world case study where we assess the ability of the system to determine the pollen make-up of samples of New Zealand honey. In addition to the Classifynder’s native neural network classifier, we have evaluated linear discriminant, support vector machine, decision tree and random forest classifiers on these data with encouraging results. Our hope is that our findings will help enhance the performance of future releases of the Classifynder and other systems for accelerating the acquisition and analysis of pollen samples.

A comparison of classification algorithms within the Classifynder pollen imaging system

We describe an investigation into how Massey University's Pollen Classifynder can accelerate the understanding of pollen and its role in nature. The Classifynder is an imaging microscopy system that can locate, image and classify slide based pollen samples. Given the laboriousness of purely manual image acquisition and identification it is vital to exploit assistive technologies like the Classifynder to enable acquisition and analysis of pollen samples. It is also vital that we understand the strengths and limitations of automated systems so that they can be used (and improved) to compliment the strengths and weaknesses of human analysts to the greatest extent possible. This article reviews some of our experiences with the Classifynder system and our exploration of alternative classifier models to enhance both accuracy and interpretability. Our experiments in the pollen analysis problem domain have been based on samples from the Australian National University's pollen reference collection (2890 grains, 15 species) and images bundled with the Classifynder system (400 grains, 4 species). These samples have been represented using the Classifynder image feature set. In addition to the Classifynder's native neural network classifier, we have evaluated linear discriminant, support vector machine, decision tree and random forest classifiers on these data with encouraging results. Our hope is that our findings will help enhance the performance of future releases of the Classifynder and other systems for accelerating the acquisition and analysis of pollen samples. © 2013 AIP Publishing LLC.

The Macroecology of Airborne Pollen in Australian and New Zealand Urban Areas

he composition and relative abundance of airborne pollen in urban areas of Australia and New Zealand are strongly influenced by geographical location, climate and land use. There is mounting evidence that the diversity and quality of airborne pollen is substantially modified by climate change and land-use yet there are insufficient data to project the future nature of these changes. Our study highlights the need for long-term aerobiological monitoring in Australian and New Zealand urban areas in a systematic, standardised, and sustained way, and provides a framework for targeting the most clinically significant taxa in terms of abundance, allergenic effects and public health burden.

Trans-disciplinary research in synthesis of grass pollen aerobiology and its importance for respiratory health in Australasia

Grass pollen is a major trigger for allergic rhinitis and asthma, yet little is known about the timing and levels of human exposure to airborne grass pollen across Australasian urban environments. The relationships between environmental aeroallergen exposure and allergic respiratory disease bridge the fields of ecology, aerobiology, geospatial science and public health. The Australian Aerobiology Working Group comprised of experts in botany, palynology, biogeography, climate change science, plant genetics, biostatistics, ecology, pollen allergy, public and environmental health, and medicine, was established to systematically source, collate and analyse atmospheric pollen concentration data from 11 Australian and six New Zealand sites. Following two week-long workshops, post-workshop evaluations were conducted to reflect upon the utility of this analysis and synthesis approach to address complex multidisciplinary questions. This Working Group described i) a biogeographically dependent variation in airborne pollen diversity, ii) a latitudinal gradient in the timing, duration and number of peaks of the grass pollen season, and iii) the emergence of new methodologies based on trans-disciplinary synthesis of aerobiology and remote sensing data. Challenges included resolving methodological variations between pollen monitoring sites and temporal variations in pollen datasets. Other challenges included “marrying” ecosystem and health sciences and reconciling divergent expert opinion. The Australian Aerobiology Working Group facilitated knowledge transfer between diverse scientific disciplines, mentored students and early career scientists, and provided an uninterrupted collaborative opportunity to focus on a unifying problem globally. The Working Group provided a platform to optimise the value of large existing ecological datasets that have importance for human respiratory health and ecosystems research. Compilation of current knowledge of Australasian pollen aerobiology is a critical first step towards the management of exposure to pollen in patients with allergic disease and provides a basis from which the future impacts of climate change on pollen distribution can be assessed and monitored.

Grass pollen allergens

Pollens from diverse grass plants are main contributors to seasonal inhalant allergies worldwide. Grass group 1 and 5 allergens represent highly cross-reactive and potent major allergens, group 5 present only in temperate climate grasses (Pooideae). Depending on climate and region, global sensitization rates to grass pollen vary between 1% to 30% of the general population,. Strong evidence supports specific immunotherapy with grass pollen extracts.

Specific IgE recognition of pollen allergens from subtropic grasses in patients from the subtropics

Background Pollens of subtropical grasses, Bahia (Paspalum notatum), Johnson (Sorghum halepense), and Bermuda (Cynodon dactylon), are common causes of respiratory allergies in subtropical regions worldwide. Objective To evaluate IgE cross-reactivity of grass pollen (GP) found in subtropical and temperate areas. Methods Case and control serum samples from 83 individuals from the subtropical region of Queensland were tested for IgE reactivity with GP extracts by enzyme-linked immunosorbent assay. A randomly sampled subset of 21 serum samples from patients with subtropical GP allergy were examined by ImmunoCAP and cross-inhibition assays. Results Fifty-four patients with allergic rhinitis and GP allergy had higher IgE reactivity with P notatum and C dactylon than with a mixture of 5 temperate GPs. For 90% of 21 GP allergic serum samples, P notatum, S halepense, or C dactylon specific IgE concentrations were higher than temperate GP specific IgE, and GP specific IgE had higher correlations of subtropical GP (r = 0.771-0.950) than temperate GP (r = 0.317-0.677). In most patients (71%-100%), IgE with P notatum, S halepense, or C dactylon GPs was inhibited better by subtropical GP than temperate GP. When the temperate GP mixture achieved 50% inhibition of IgE with subtropical GP, there was a 39- to 67-fold difference in concentrations giving 50% inhibition and significant differences in maximum inhibition for S halepense and P notatum GP relative to temperate GP. Conclusion Patients living in a subtropical region had species specific IgE recognition of subtropical GP. Most GP allergic patients in Queensland would benefit from allergen specific immunotherapy with a standardized content of subtropical GP allergens.

Regional and seasonal variation in airborne grass pollen levels between cities of Australia and New Zealand

Although grass pollen is widely regarded as the major outdoor aeroallergen source in Australia and New Zealand (NZ), no assemblage of airborne pollen data for the region has been previously compiled. Grass pollen count data collected at 14 urban sites in Australia and NZ over periods ranging from 1 to 17 years were acquired, assembled and compared, revealing considerable spatiotemporal variability. Although direct comparison between these data is problematic due to methodological differences between monitoring sites, the following patterns are apparent. Grass pollen seasons tended to have more than one peak from tropics to latitudes of 37°S and single peaks at sites south of this latitude. A longer grass pollen season was therefore found at sites below 37°S, driven by later seasonal end dates for grass growth and flowering. Daily pollen counts increased with latitude; subtropical regions had seasons of both high intensity and long duration. At higher latitude sites, the single springtime grass pollen peak is potentially due to a cooler growing season and a predominance of pollen from C3 grasses. The multiple peaks at lower latitude sites may be due to a warmer season and the predominance of pollen from C4 grasses. Prevalence and duration of seasonal allergies may reflect the differing pollen seasons across Australia and NZ. It must be emphasized that these findings are tentative due to limitations in the available data, reinforcing the need to implement standardized pollen-monitoring methods across Australasia. Furthermore, spatiotemporal differences in grass pollen counts indicate that local, current, standardized pollen monitoring would assist with the management of pollen allergen exposure for patients at risk of allergic rhinitis and asthma. © 2015 Springer Science+Business Media Dordrecht

Do human rhinovirus infections and food allergy modify grass pollen-induced asthma hospital admissions in children?

Asthma prevalence in children has remained relatively constant in many Western countries, but hospital admissions for younger age groups have increased over time.1 Although the role of outdoor aeroallergens as triggers for asthma exacerbations requiring hospitalization in children and adolescents is complex, there is evidence that increasing concentrations of grass pollen are associated with an increased risk of asthma exacerbations in children.2 Human rhinovirus (HRV) infections are implicated in most of the serious asthma exacerbations in school-age children.3 In previous research, HRV infections and aeroallergen exposure have usually been studied independently. To our knowledge, only 1 study has examined interactions between these 2 factors,4 but lack of power prevented any meaningful interpretation...

Total transcriptome, proteome, and allergome of Johnson grass pollen, which is important for allergic rhinitis in subtropical regions

Background Genomic data are lacking for many allergen sources. To circumvent this limitation, we implemented a strategy to reveal the repertoire of pollen allergens of a grass with clinical importance in subtropical regions, where an increasing proportion of the world's population resides. Objective We sought to identify and immunologically characterize the allergenic components of the Panicoideae Johnson grass pollen (JGP; Sorghum halepense). Methods The total pollen transcriptome, proteome, and allergome of JGP were documented. Serum IgE reactivities with pollen and purified allergens were assessed in 64 patients with grass pollen allergy from a subtropical region. Results Purified Sor h 1 and Sor h 13 were identified as clinically important allergen components of JGP with serum IgE reactivity in 49 (76%) and 28 (43.8%), respectively, of patients with grass pollen allergy. Within whole JGP, multiple cDNA transcripts and peptide spectra belonging to grass pollen allergen families 1, 2, 4, 7, 11, 12, 13, and 25 were identified. Pollen allergens restricted to subtropical grasses (groups 22-24) were also present within the JGP transcriptome and proteome. Mass spectrometry confirmed the IgE-reactive components of JGP included isoforms of Sor h 1, Sor h 2, Sor h 13, and Sor h 23. Conclusion Our integrated molecular approach revealed qualitative differences between the allergenic components of JGP and temperate grass pollens. Knowledge of these newly identified allergens has the potential to improve specific diagnosis and allergen immunotherapy treatment for patients with grass pollen allergy in subtropical regions and reduce the burden of allergic respiratory disease globally.

Differences in grass pollen allergen exposure across Australia

Objective Allergic rhinitis and allergic asthma are important chronic diseases posing serious public health issues in Australia with associated medical, economic, and societal burdens. Pollen are significant sources of clinically relevant outdoor aeroallergens, recognised as both a major trigger for, and cause of, allergic respiratory diseases. This study aimed to provide a national, and indeed international, perspective on the state of Australian pollen data using a large representative sample. Methods Atmospheric grass pollen concentration is examined over a number of years within the period 1995 to 2013 for Brisbane, Canberra, Darwin, Hobart, Melbourne, and Sydney, including determination of the clinical' grass pollen season and grass pollen peak. Results The results of this study describe, for the first time, a striking spatial and temporal variability in grass pollen seasons in Australia, with important implications for clinicians and public health professionals, and the Australian grass pollen-allergic community. Conclusions These results demonstrate that static pollen calendars are of limited utility and in some cases misleading. This study also highlights significant deficiencies and limitations in the existing Australian pollen monitoring and data. Implications: Establishment of an Australian national pollen monitoring network would help facilitate advances in the clinical and public health management of the millions of Australians with asthma and allergic rhinitis.

An immunodiagnostic assay for quantitation of specific ige to the major pollen allergen component, pas n 1, of the subtropical bahia grass

Background Pollens of the Panicoideae subfamily of grasses including Bahia (Paspalum notatum) are important allergen sources in subtropical regions of the world. An assay for specific IgE to the major molecular allergenic component, Pas n 1, of Bahia grass pollen (BaGP) would have immunodiagnostic utility for patients with pollen allergy in these regions. Methods Biotinylated Pas n 1 purified from BaGP was coated onto streptavidin ImmunoCAPs. Subjects were assessed by clinical history of allergic rhinitis and skin prick test (SPT) to aeroallergens. Serum total, BaGP-specific and Pas n 1-specific IgE were measured. Results: Pas n 1 IgE concentrations were highly correlated with BaGP SPT (r = 0.795, p < 0.0001) and BaGP IgE (r = 0.915, p < 0.0001). At 0.23 kU/l Pas n 1 IgE, the diagnostic sensitivity (92.4%) and specificity (93.1%) for the detection of BaGP allergy was high (area under receiver operator curve 0.960, p < 0.0001). The median concentrations of Pas n 1 IgE in non-Atopic subjects (0.01 kU/l, n = 67) and those with other allergies (0.02 kU/l, n = 59) showed no inter-group difference, whilst grass pollen-Allergic patients with allergic rhinitis showed elevated Pas n 1 IgE (6.71 kU/l, n = 182, p < 0.0001). The inter-Assay coefficient of variation for the BaGP-Allergic serum pool was 6.92%. Conclusions Pas n 1 IgE appears to account for most of the BaGP-specific IgE. This molecular component immunoassay for Pas n 1 IgE has potential utility to improve the sensitivity and accuracy of diagnosis of BaGP allergy for patients in subtropical regions.

Grass pollen allergens globally: The contribution of subtropical grasses to burden of allergic respiratory diseases

Grass pollens of the temperate (Pooideae) subfamily and subtropical subfamilies of grasses are major aeroallergen sources worldwide. The subtropical Chloridoideae (e.g. Cynodon dactylon; Bermuda grass) and Panicoideae (e.g. Paspalum notatum; Bahia grass) species are abundant in parts of Africa, India, Asia, Australia and the Americas, where a large and increasing proportion of the world's population abide. These grasses are phylogenetically and ecologically distinct from temperate grasses. With the advent of global warming, it is conceivable that the geographic distribution of subtropical grasses and the contribution of their pollen to the burden of allergic rhinitis and asthma will increase. This review aims to provide a comprehensive synthesis of the current global knowledge of (i) regional variation in allergic sensitivity to subtropical grass pollens, (ii) molecular allergenic components of subtropical grass pollens and (iii) allergic responses to subtropical grass pollen allergens in relevant populations. Patients from subtropical regions of the world show higher allergic sensitivity to grass pollens of Chloridoideae and Panicoideae grasses, than to temperate grass pollens. The group 1 allergens are amongst the allergen components of subtropical grass pollens, but the group 5 allergens, by which temperate grass pollen extracts are standardized for allergen content, appear to be absent from both subfamilies of subtropical grasses. Whilst there are shared allergenic components and antigenic determinants, there are additional clinically relevant subfamily-specific differences, at T- and B-cell levels, between pollen allergens of subtropical and temperate grasses. Differential immune recognition of subtropical grass pollens is likely to impact upon the efficacy of allergen immunotherapy of patients who are primarily sensitized to subtropical grass pollens. The literature reviewed herein highlights the clinical need to standardize allergen preparations for both types of subtropical grass pollens to achieve optimal diagnosis and treatment of patients with allergic respiratory disease in subtropical regions of the world. © 2014 John Wiley & Sons Ltd.