Grass pollen exposure is associated with higher readmission rates for pediatric asthma.

BACKGROUND: Pediatric asthma hospital readmission is a burden on the individual and costly for Australian hospitals. Grass pollen's role, a known trigger for asthma admissions, is unexamined in readmissions. We examined the association between grass pollen and pediatric asthma readmission. METHODS: The Victorian Admitted Episodes Dataset was used to identify all primary admissions with a principal diagnosis of asthma in children aged 2-18 years between 1997 and 2009. Readmissions were defined as subsequent admissions within 28 days of index admission discharge. Generalized additive models were used to assess associations between readmission, grass pollen season, and daily grass pollen counts, lagged and cumulative. Models were further stratified by sex and age group. RESULTS: Mean daily readmission was higher during grass pollen season than other times of the year, incidence rate ratio (IRR) 1.44 (95% CI, 1.03, 2.02) and for children aged 2-5 years, IRR 1.99 (1.26, 3.14). Same day grass pollen was nonlinearly associated with daily readmission for the 13-18 age group between 110 and 256 grains/m3 , p < .01. Lag 2 grass pollen was nonlinearly associated with daily readmissions overall (p = .03), boys (p = .01), and younger age groups 2-5 (p = .02) and 6-12 (p < .001). CONCLUSIONS: Grass pollen exposure was associated with higher readmission rates for pediatric asthma. Treatment plans prior to discharge could be implemented to reduce the likelihood of readmission by younger children during the pollen season.

The AusPollen partnership project: Allergenic airborne grass pollen seasonality and magnitude across temperate and subtropical eastern Australia, 2016-2020.

BACKGROUND: Allergic rhinitis affects half a billion people globally, including a fifth of the Australian population. As the foremost outdoor allergen source, ambient grass pollen exposure is likely to be altered by climate change. The AusPollen Partnership aimed to standardize pollen monitoring and examine broad-scale biogeographical and meteorological factors influencing interannual variation in seasonality of grass pollen aerobiology in Australia. METHODS: Daily airborne grass and other pollen concentrations in four eastern Australian cities separated by over 1700 km, were simultaneously monitored using Hirst-style samplers following the Australian Interim Pollen and Spore Monitoring Standard and Protocols over four seasons from 2016 to 2020. The grass seasonal pollen integral was determined. Gridded rainfall, temperature, and satellite-derived grassland sources up to 100 km from the monitoring site were analysed. RESULTS: The complexity of grass pollen seasons was related to latitude with multiple major summer-autumn peaks in Brisbane, major spring and minor summer peaks in Sydney and Canberra, and single major spring peaks occurring in Melbourne. The subtropical site of Brisbane showed a higher proportion of grass out of total pollen than more temperate sites. The magnitude of the grass seasonal pollen integral was correlated with pasture greenness, rainfall and number of days over 30 °C, preceding and within the season, up to 100 km radii from monitoring sites. CONCLUSIONS: Interannual fluctuations in Australian grass pollen season magnitude are strongly influenced by regional biogeography and both pre- and in-season weather. This first continental scale, Southern Hemisphere standardized aerobiology dataset forms the basis to track shifts in pollen seasonality, biodiversity and impacts on allergic respiratory diseases.

The association between outdoor allergens - pollen, fungal spore season and high asthma admission days in children and adolescents.

Periods when asthma admissions peaks have serious implications for asthma sufferers and hospitals. We assessed the association between aeroallergen exposure and childhood asthma peak periods during two grass pollen seasons using the Melbourne Air Pollen Children and Adolescent Health (MAPCAH) study conducted in Melbourne, Australia. Two peak periods were identified. Effect modifications by atopy and sex were considered. All pollen 2 days prior was associated with increased odds of these peak periods. Same day fungal spores, but not pollen, were important. Grass at lag 2 was associated with increased odds 1.03 (95%CI 1.01, 1.05) as was the same day Alternaria 1.02 (1.00, 1.04) per spore/m3 for boys. In addition to pollen, fungal spores particularly Alternaria may result in days of high exacerbations during pollen seasons. Further guidance is needed to better prepare families/carers with information about the increased risk of asthma attacks in children prior to pollen seasons.

Thunderstorm asthma in seasonal allergic rhinitis: The TAISAR study.

BACKGROUND: Asthma epidemics associated with thunderstorms have had catastrophic effects on individuals and emergency services. Seasonal allergic rhinitis (SAR) is present in the vast majority of people who develop thunderstorm asthma (TA), but there is little evidence regarding risk factors for TA among the SAR population. OBJECTIVE: We sought to identify risk factors for a history of TA and hospital presentation in a cohort of individuals with SAR. METHODS: This multicenter study recruited adults from Melbourne, Australia, with a past diagnosis of TA and/or self-reported SAR. Clinical information, spirometry results, white blood cell count, ryegrass pollen-specific (RGP-sp) IgE concentration, and fractional exhaled nitric oxide were measured to identify risk factors for a history of TA in individuals with SAR. RESULTS: From a total of 228 individuals with SAR, 35% (80 of 228) reported SAR only (the I-SAR group), 37% (84 of 228) reported TA symptoms but had not attended hospital for treatment (the O-TA group), and 28% (64 of 228) had presented to the hospital for TA (the H-TA group). All patients in the H-TA group reported a previous asthma diagnosis. Logistic regression analysis of factors associated with O-TA and H-TA indicated that lower FEV1 value and an Asthma Control Questionnaire score higher than 1.5 were associated with H-TA. Higher blood RGP-sp IgE concentration, eosinophil counts, and fractional exhaled nitric oxide level were significantly associated with both O-TA and H-TA. Receiver operating curve analysis showed an RGP-sp IgE concentration higher than 10.1 kU/L and a prebronchodilator FEV1 value of 90% or lower to be biomarkers of increased H-TA risk. CONCLUSION: Clinical tests can identify risk of a history of TA in individuals with SAR and thereby inform patient-specific treatment recommendations.

Atmospheric modelling of grass pollen rupturing mechanisms for thunderstorm asthma prediction.

The world's most severe thunderstorm asthma event occurred in Melbourne, Australia on 21 November 2016, coinciding with the peak of the grass pollen season. The aetiological role of thunderstorms in these events is thought to cause pollen to rupture in high humidity conditions, releasing large numbers of sub-pollen particles (SPPs) with sizes very easily inhaled deep into the lungs. The humidity hypothesis was implemented into a three-dimensional atmospheric model and driven by inputs from three meteorological models. However, the mechanism could not explain how the Melbourne event occurred as relative humidity was very low throughout the atmosphere, and most available grass pollen remained within 40 m of the surface. Our tests showed humidity induced rupturing occurred frequently at other times and would likely lead to recurrent false alarms if used in a predictive capacity. We used the model to investigate a range of other possible pollen rupturing mechanisms which could have produced high concentrations of SPPs in the atmosphere during the storm. The mechanisms studied involve mechanical friction from wind gusts, electrical build up and discharge incurred during conditions of low relative humidity, and lightning strikes. Our results suggest that these mechanisms likely operated in tandem with one another, but the lightning method was the only mechanism to generate a pattern in SPPs following the path of the storm. If humidity induced rupturing cannot explain the 2016 Melbourne event, then new targeted laboratory studies of alternative pollen rupture mechanisms would be of considerable value to help constrain the parameterisation of the pollen rupturing process.

Is short-term exposure to grass pollen adversely associated with lung function and airway inflammation in the community?

BACKGROUND: The association between grass pollen exposure and early markers of asthma exacerbations such as lung function changes and increase in airway inflammation is limited. We investigated the associations between short-term grass pollen exposure and lung function and airway inflammation in a community-based sample, and whether any such associations were modified by current asthma, current hay fever, pollen sensitization, age, and other environmental factors. METHODS: Cross-sectional and short-term analyses of data from the Melbourne Atopy Cohort Study (MACS) participants (n = 936). Lung function was assessed using spirometry. Airway inflammation was assessed by fractional exhaled nitric oxide (FeNO) and exhaled breath condensate pH and nitrogen oxides (NOx). Daily pollen counts were collected using a volumetric spore trap. The associations were examined by linear regression. RESULTS: Higher ambient levels of grass pollen 2 days before (lag 2) were associated with lower mid-forced expiratory flow (FEF25%-75% ) and FEV1 /FVC ratio (Coef. [95% CI] = -119 [-226, -11] mL/s and -1.0 [-3.0, -0.03] %, respectively) and also 3 days before (lag 3). Increased levels of grass pollen a day before (lag 1) were associated with increased FeNO (4.35 [-0.1, 8.7] ppb) and also at lag 2. Adverse associations between pollen and multiple outcomes were greater in adults with current asthma, hay fever, and pollen sensitization. CONCLUSION: Grass pollen exposure was associated with eosinophilic airway inflammation 1-2 days after exposure and airway obstruction 2-3 days after exposure. Adults and individuals with asthma, hay fever, and pollen sensitization may be at higher risk.

Using crowd-sourced allergic rhinitis symptom data to improve grass pollen forecasts and predict individual symptoms.

Seasonal allergic rhinitis (AR), also known as hay fever, is a common respiratory condition brought on by a range of environmental triggers. Previous work has characterised the relationships between community-level AR symptoms collected using mobile apps in two Australian cities, Canberra and Melbourne, and various environmental covariates including pollen. Here, we build on these relationships by assessing the skill of models that provide a next-day forecast of an individual's risk of developing AR and that nowcast ambient grass pollen concentrations using crowd-sourced AR symptoms as a predictor. Categorical grass pollen forecasts (low/moderate/high) were made based on binning mean daily symptom scores by corresponding categories. Models for an individual's risk were constructed by forward variable selection, considering environmental, demographic, behaviour and health-related inputs, with non-linear responses permitted. Proportional-odds logistic regression was then applied with the variables selected, modelling the symptom scores on their original five-point scale. AR symptom-based estimates of today's average grass pollen concentration were more accurate than those provided by two benchmark forecasting methods using various metrics for assessing accuracy. Predictions of an individual's next-day AR symptoms rated on a five-point scale were correct in 36% of cases and within one point on this scale in 82% of cases. Both outcomes were significantly better than chance. This large-scale AR symptoms measurement program shows that crowd-sourced symptom scores can be used to predict the daily average grass pollen concentration, as well as provide a personalised AR forecast.

Crowd-sourced allergic rhinitis symptom data: The influence of environmental and demographic factors.

Allergic Rhinitis (AR) affects over half a billion people worldwide with an estimated prevalence of 1 in 5 individuals in developed countries. Although ambient pollen exposure is a causal factor in AR, the symptom-exposure relationship is typically not studied in the broader community but in small, well-characterised cohorts drawn from clinical populations. To identify relationships between AR symptoms in the community and a range of environmental factors, we used a database containing over 96,000 symptom score reports collected over a 3-year period (2014-2016) through freely available smartphone apps released in two Australian cities, Melbourne and Canberra. Ambient pollen levels and symptom scores were strongly related, with grass pollen explaining most of the symptom variation. Other factors correlated with higher symptom scores included temperature (R > 0.73) and wind speed (R > 0.75). In general, worse symptom scores were reported by younger participants, women, and those who had taken medication for AR in the preceding 24 h. The strength of this relationship varied between the two cities. Smartphone-based symptom surveys offer a cost-effective means of studying real-world risk factors for AR in a broader 'extra-clinical' population.

Intensive care implications of epidemic thunderstorm asthma.

OBJECTIVE: To investigate the environmental precipitants, treatment and outcome of critically ill patients affected by the largest and most lethal reported epidemic of thunderstorm asthma. DESIGN, SETTING AND PARTICIPANTS: Retrospective multicentre observational study. Meteorological, airborne particulate and pollen data, and a case series of 35 patients admitted to 15 intensive care units (ICUs) due to the thunderstorm asthma event of 21-22 November 2016, in Victoria, Australia, were analysed and compared with 1062 total ICU-admitted Australian patients with asthma in 2016. MAIN OUTCOME MEASURES: Characteristics and outcomes of total ICU versus patients with thunderstorm asthma, the association between airborne particulate counts and storm arrival, and ICU resource utilisation. RESULTS: All 35 patients had an asthma diagnosis; 13 (37%) had a cardiac or respiratory arrest, five (14%) died. Compared with total Australian ICU-admitted patients with asthma in 2016, patients with thunderstorm asthma had a higher mortality (15% v 1.3%, P < 0.001), were more likely to be male (63% v 34%, P < 0.001), to be mechanically ventilated, and had shorter ICU length of stay in survivors (median, 31.8 hours [interquartile range (IQR), 14.8-43.6 hours] v 40.7 hours [IQR, 22.3-75.1 hours]; P = 0.025). Patients with cardiac arrest were more likely to be born in Asian or subcontinental countries (5/10 [50%] v 4/25 [16%]; relative risk, 3.13; 95% CI, 1.05-9.31). A temporal link was demonstrated between airborne particulate counts and arrival of the storm. The event used 15% of the public ICU beds in the region. CONCLUSION: Arrival of a triggering storm is associated with an increase in respirable airborne particles. Affected critically ill patients are young, have a high mortality, a short duration of bronchospasm, and a prior diagnosis of asthma is common.

The Melbourne epidemic thunderstorm asthma event 2016: an investigation of environmental triggers, effect on health services, and patient risk factors.

BACKGROUND: A multidisciplinary collaboration investigated the world's largest, most catastrophic epidemic thunderstorm asthma event that took place in Melbourne, Australia, on Nov 21, 2016, to inform mechanisms and preventive strategies. METHODS: Meteorological and airborne pollen data, satellite-derived vegetation index, ambulance callouts, emergency department presentations, and data on hospital admissions for Nov 21, 2016, as well as leading up to and following the event were collected between Nov 21, 2016, and March 31, 2017, and analysed. We contacted patients who presented during the epidemic thunderstorm asthma event at eight metropolitan health services (each including up to three hospitals) via telephone questionnaire to determine patient characteristics, and investigated outcomes of intensive care unit (ICU) admissions. FINDINGS: Grass pollen concentrations on Nov 21, 2016, were extremely high (>100 grains/m3). At 1800 AEDT, a gust front crossed Melbourne, plunging temperatures 10°C, raising humidity above 70%, and concentrating particulate matter. Within 30 h, there were 3365 (672%) excess respiratory-related presentations to emergency departments, and 476 (992%) excess asthma-related admissions to hospital, especially individuals of Indian or Sri Lankan birth (10% vs 1%, p<0·0001) and south-east Asian birth (8% vs 1%, p<0·0001) compared with previous 3 years. Questionnaire data from 1435 (64%) of 2248 emergency department presentations showed a mean age of 32·0 years (SD 18·6), 56% of whom were male. Only 28% had current doctor-diagnosed asthma. 39% of the presentations were of Asian or Indian ethnicity (25% of the Melbourne population were of this ethnicity according to the 2016 census, relative risk [RR] 1·93, 95% CI 1·74-2·15, p <0·0001). Of ten individuals who died, six were Asian or Indian (RR 4·54, 95% CI 1·28-16·09; p=0·01). 35 individuals were admitted to an intensive care unit, all had asthma, 12 took inhaled preventers, and five died. INTERPRETATION: Convergent environmental factors triggered a thunderstorm asthma epidemic of unprecedented magnitude, tempo, and geographical range and severity on Nov 21, 2016, creating a new benchmark for emergency and health service escalation. Asian or Indian ethnicity and current doctor-diagnosed asthma portended life-threatening exacerbations such as those requiring admission to an ICU. Overall, the findings provide important public health lessons applicable to future event forecasting, health care response coordination, protection of at-risk populations, and medical management of epidemic thunderstorm asthma. FUNDING: None.

Seasonal asthma in Melbourne, Australia, and some observations on the occurrence of thunderstorm asthma and its predictability.

We examine the seasonality of asthma-related hospital admissions in Melbourne, Australia, in particular the contribution and predictability of episodic thunderstorm asthma. Using a time-series ecological approach based on asthma admissions to Melbourne metropolitan hospitals, we identified seasonal peaks in asthma admissions that were centred in late February, June and mid-November. These peaks were most likely due to the return to school, winter viral infections and seasonal allergies, respectively. We performed non-linear statistical regression to predict daily admission rates as functions of the seasonal cycle, weather conditions, reported thunderstorms, pollen counts and air quality. Important predictor variables were the seasonal cycle and mean relative humidity in the preceding two weeks, with higher humidity associated with higher asthma admissions. Although various attempts were made to model asthma admissions, none of the models explained substantially more variation above that associated with the annual cycle. We also identified a list of high asthma admissions days (HAADs). Most HAADs fell in the late-February return-to-school peak and the November allergy peak, with the latter containing the greatest number of daily admissions. Many HAADs in the spring allergy peak may represent episodes of thunderstorm asthma, as they were associated with rainfall, thunderstorms, high ambient grass pollen levels and high humidity, a finding that suggests thunderstorm asthma is a recurrent phenomenon in Melbourne that occurs roughly once per five years. The rarity of thunderstorm asthma events makes prediction challenging, underscoring the importance of maintaining high standards of asthma management, both for patients and health professionals, especially during late spring and early summer.

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.

On the causes of variability in amounts of airborne grass pollen in Melbourne, Australia.

In Melbourne, Australia, airborne grass pollen is the predominant cause of hay fever (seasonal rhinitis) during late spring and early summer, with levels of airborne grass pollen also influencing hospital admissions for asthma. In order to improve predictions of conditions that are potentially hazardous to susceptible individuals, we have sought to better understand the causes of diurnal, intra-seasonal and inter-seasonal variability of atmospheric grass pollen concentrations (APC) by analysing grass pollen count data for Melbourne for 16 grass pollen seasons from 1991 to 2008 (except 1994 and 1995). Some of notable features identified in this analysis were that on days when either extreme (>100 pollen grains m(-3)) or high (50-100 pollen grains m(-3)) levels of grass pollen were recorded the winds were of continental origin. In contrast, on days with a low (<20 pollen grains m(-3)) concentration of grass pollen, winds were of maritime origin. On extreme and high grass pollen days, a peak in APC occurred on average around 1730 hours, probably due to a reduction in surface boundary layer turbulence. The sum of daily APC for each grass pollen season was highly correlated (r = 0.79) with spring rainfall in Melbourne for that year, with about 60% of a declining linear trend across the study period being attributable to a reduction of meat cattle and sheep (and hence grazing land) in rural areas around Melbourne. Finally, all of the ten extreme pollen events (3 days or more with APC > 100 pollen grains m(-3)) during the study period were characterised by an average downward vertical wind anomaly in the surface boundary layer over Melbourne. Together these findings form a basis for a fine resolution atmospheric general circulation model for grass pollen in Melbourne's air that can be used to predict daily (and hourly) APC. This information will be useful to those sectors of Melbourne's population that suffer from allergic problems.

Aberrant cell expansion in the elongation mutants of barley.

The elongation (elo) mutants of barley (Hordeum vulgare cv 'Himalaya') are a class of dwarf plants with defects affecting cell expansion. The phenotypes of mutants in three of the elo loci (elo1, elo2 and elo3) are recessive to the wild-type allele, and the mutations at elo-4 and elo-5 are semi-dominant. Allelism tests showed that elo1, elo2 and elo3 were at separate loci, and mapping data indicated that elo-5 was possibly allelic to either elo1 or elo2. A phenotype common to all elo mutants was the presence of short, radially swollen cells on the leaf epidermis, indicating a defect in longitudinal cell expansion. In three of the mutants, elo1, elo3 and elo5, this was accompanied by a twisting growth habit. Two of the mutations, elo2 and elo-5, affected cell division, with aberrant periclinal cell division resulting in the formation of increased cell layers in the leaf epidermis of elo2 and elo-5 homozygotes and in the aleurone layer of elo2 grains. Misplaced anticlinal divisions also occurred in the elo-5 leaf epidermis. Leaf cell walls of all elo lines contained less cellulose than the wild- type, and the cortical microtubules in elongating root epidermal cells in some elo lines were more randomly oriented than in the wild-type, consistent with the presence of radially swollen cells. We discuss possible functions for the Elo genes in primary cell wall synthesis.

Analysis of the Oryza sativa plasma membrane proteome using combined protein and peptide fractionation approaches in conjunction with mass spectrometry.

To identify integral and peripheral plasma membrane (PM) proteins from Oryza sativa (rice), highly enriched PM fractions from rice suspension cultured cells were analyzed using two complementary approaches. The PM was enriched using aqueous two-phase partitioning and high pH carbonate washing to remove soluble, contaminating proteins and characterized using enzymatic and immunological analyses. Proteins from the carbonate-washed PM (WPM) were analyzed by either one-dimensional gel electrophoresis (1D-SDS-PAGE) followed by tryptic proteolysis or proteolysis followed by strong cation exchange liquid chromatography (LC) with subsequent analysis of the tryptic peptides by LC-MS/MS (termed Gel-LC-MS/MS and 2D-LC-MS/MS, respectively). Combining the results of these two approaches, 438 proteins were identified on the basis of two or more matching peptides, and a further 367 proteins were identified on the basis of single peptide matches after data analysis with two independent search algorithms. Of these 805 proteins, 350 were predicted to be PM or PM-associated proteins. Four hundred and twenty-five proteins (53%) were predicted to be integrally associated with a membrane, via either one or many (up to 16) transmembrane domains, a GPI-anchor, or membrane-spanning beta-barrels. Approximately 80% of the 805 identified proteins were assigned a predicted function, based on similarity to proteins of known function or the presence of functional domains. Proteins involved in PM-related activities such as signaling (21% of the 805 proteins), transporters and ATPases (14%), and cellular trafficking (8%), such as via vesicles involved in endo- and exocytosis, were identified. Proteins that are involved in cell wall biosynthesis were also identified (5%) and included three cellulose synthase (CESA) proteins, a cellulose synthase-like D (CSLD) protein, cellulases, and several callose synthases. Approximately 20% of the proteins identified in this study remained functionally unclassified despite being predicted to be membrane proteins.

Cellulose synthase-like CslF genes mediate the synthesis of cell wall (1,3;1,4)-beta-D-glucans.

A characteristic feature of grasses and commercially important cereals is the presence of (1,3;1,4)-beta-d-glucans in their cell walls. We have used comparative genomics to link a major quantitative trait locus for (1,3;1,4)-beta-d-glucan content in barley grain to a cluster of cellulose synthase-like CslF genes in rice. After insertion of rice CslF genes into Arabidopsis, we detected (1,3;1,4)-beta-d-glucan in walls of transgenic plants using specific monoclonal antibodies and enzymatic analysis. Because wild-type Arabidopsis does not contain CslF genes or have (1,3;1,4)-beta-d-glucans in its walls, these experiments provide direct, gain-of-function evidence for the participation of rice CslF genes in (1,3;1,4)-beta-d-glucan biosynthesis.

Temporal and spatial appearance of wall polysaccharides during cellularization of barley (Hordeum vulgare) endosperm.

Barley endosperm begins development as a syncytium where numerous nuclei line the perimeter of a large vacuolated central cell. Between 3 and 6 days after pollination (DAP) the multinucleate syncytium is cellularized by the centripetal synthesis of cell walls at the interfaces of nuclear cytoplasmic domains between individual nuclei. Here we report the temporal and spatial appearance of key polysaccharides in the cell walls of early developing endosperm of barley, prior to aleurone differentiation. Flowering spikes of barley plants grown under controlled glasshouse conditions were hand-pollinated and the developing grains collected from 3 to 8 DAP. Barley endosperm development was followed at the light and electron microscope levels with monoclonal antibodies specific for (1-->3)-beta-D: -glucan (callose), (1-->3,1-->4)-beta-D: -glucan, hetero-(1-->4)-beta-D: -mannans, arabino-(1-->4)-beta-D: -xylans, arabinogalactan-proteins (AGPs) and with the enzyme, cellobiohydrolase II, to detect (1-->4)-beta-D: -glucan (cellulose). Callose and cellulose were present in the first formed cell walls between 3 and 4 DAP. However, the presence of callose in the endosperm walls was transient and at 6 DAP was only detected in collars surrounding plasmodesmata. (1-->3,1-->4)-beta-D: -Glucan was not deposited in the developing cell walls until approximately 5 DAP and hetero-(1-->4)-beta-D: -mannans followed at 6 DAP. Deposition of AGPs and arabinoxylan in the wall began at 7 and 8 DAP, respectively. For arabinoxylans, there is a possibility that they are deposited earlier in a highly substituted form that is inaccessible to the antibody. Arabinoxylan and heteromannan were also detected in Golgi and associated vesicles in the cytoplasm. In contrast, (1-->3,1-->4)-beta-D: -glucan was not detected in the cytoplasm in endosperm cells; similar results were obtained for coleoptile and suspension cultured cells.