Occurrence of mono- or disaccharides and polysaccharide reserves in mature pollen grains

 Pollen from 13 species of gymnosperms and angiosperms was studied for soluble and insoluble carbohydrates at dispersal. Starch reserves stored during pollen development give rise to carbohydrates at maturity. Combinations of different types of carbohydrates in mature pollen may depend on the extent of starch hydrolysis. An inverse relationship was found between the extent of starch hydrolysis and sucrose content. If the starch was scarcely de-polymerized, the cytoplasm had very low levels of soluble sugars and none of the periodic acid-Schiff (PAS)-positive material as found in pollen not subject to high dehydration (Cucurbita pepo L., Zea mays L.). After total or partial starch hydrolysis, insoluble PAS-positive oligo/polysaccharides were found in the cytoplasm associated with much soluble sugar, and the pollen grains were dehydrated at dispersal as in Typha latifolia L., Chamaerops humilis L., Trachycarpus excelsa Wendl., and other specimens. Intermediate levels of starch and soluble sugars, together with cytoplasmic PAS-positive material, characterized species with dehydrated pollen such as Pinus halepensis Miller. Carbohydrates may be related to pollen longevity, which largely depends on the abundance of sucrose, which is known to protect membrane integrity. The relationship between PAS-positive material and pollen viability is unclear at present. Received: 30 July 1996 / Revision accepted: 18 December 1996     

Ripe pollen carbohydrate changes in Trachycarpus fortunei: the effect of relative humidity

Pollen of the palm Trachycarpus fortunei was kept at 25°C and relative humidities (RH) of 20, 55 and 98%. Changes in viability, water content and carbohydrates were measured over 2–17 days. Water content remained almost constant at 20 and 50% RH and increased dramatically at 98%. Pollen viability and germination rate remained almost constant over 14 days at 20% RH and decreased to about 2% after 7–9 days at 55% and to even less at 98% RH. Although the three experimental conditions were constant, qualitative and quantitative variations in pollen carbohydrates were recorded, even after pollen had lost its viability. The quantities of mono-, di- and polysaccharides varied with the period of pollen storage at the various RH. The greatest changes in glucose, fructose and sucrose content were recorded at 55 and 98% RH. At these relative humidities, maximum glucose and fructose content and minimum sucrose content occurred at maximum water content. Starch was not present in mature pollen but appeared and peaked after 7–9 days of pollen storage at 55 and 98%. Appearance of starch coincided with an increase in pectin content. PAS-positive cytoplasmic polysaccharides showed an increasing trend at 20% RH. A relation was found between pollen viability, water content and monosaccharide content. Pollen viability and germination capacity remained high at 20% RH for 14 days. At this relative humidity, pollen water, glucose and fructose contents remained almost constant, while sucrose reached its maximum value. The fluctuations of more complex carbohydrates (starch, pectins and PAS-positive cytoplasmic polysaccharides) were less easy to interpret. Changes observed under experimental conditions could simulate processes occurring in nature during pollen presentation and dispersal.     

Pollen hydration status at dispersal: cytophysiological features and strategies

Summary The aim of this paper is to draw attention to partially hydrated pollen, namely, pollen grains having a high water content (>30%); this type of pollen is more frequent than previously thought. Various cyto-physiological strategies are used to retain water during exposure and dispersal such as cytoplasm carbohydrates; in the absence of such strategies, fast pollination must be ensured, because uncontrolled loss of water leads to pollen death. On the other hand, a state of partial hydration allows a fast tube emission (even within 3–5 min). Several methods for determining the hydration status of pollen at anthesis are proposed.     

Changes of pollen viability of ornamental plants after long-term preservation in a cryopreservation pollen bank

This study determined the changes in pollen viability of 102 species/cultivars of ornamental plants (affiliated to 32 genera of 14 families) following long-term liquid nitrogen storage in a cryopreservation pollen bank. The goal was to provide information on the safety and stability of pollen cryopreservation technology. Fresh pollen at the time of storage was used as the control, and the study examined the pollen viability of ornamental plants cryopreserved for 8, 9, or 10 years. The results show that pollen of the 102 species/cultivars in the cryopreservation pollen bank retained viability ranging from 1% to 58%, After long-term storage there were changes in viability: 11.76% (12 species/cultivars) had increased viability, 16.67% (17 species/cultivars) had stable viability, and the viability of 71.57% (73 species/cultivars) showed a decreasing trend.     

Actin and pollen tube growth

Summary Actin microfilaments (MFs) are essential for the growth of the pollen tube. Although it is well known that MFs, together with myosin, deliver the vesicles required for cell elongation, it is becoming evident that the polymerization of new actin MFs, in a process that is independent of actomyosin-dependent vesicle translocation, is also necessary for cell elongation. Herein we review the recent literature that focuses on this subject, including brief discussions of the actin-binding proteins in pollen, and their possible role in regulating actin MF activity. We promote the view that polymerization of new actin MFs polarizes the cytoplasm at the apex of the tube. This process is regulated in part by the apical calcium gradient and by different actin-binding proteins. For example, profilin binds actin monomers and gives the cell control over the initiation of polymerization. A more recently discovered actin-binding protein, villin, stimulates the formation of unipolar bundles of MFs. Villin may also respond to the apical calcium gradient, fragmenting MFs, and thus locally facilitating actin remodeling. While much remains to be discovered, it is nevertheless apparent that actin MFs play a fundamental role in controlling apical cell growth in pollen tubes.Dedicated to Professor Brian E. S. Gunning on the occasion of his 65th birthday     

Characterization of the translocator associated with pollen tube organelles

Summary In pollen tubes, the motive force of cytoplasmic streaming is assumed to be generated by the sliding of the translocator associated with cell organelles along actin filaments. In the present study, the characteristics of the translocator were studied by reconstituting the movement of pollen tube organelles along characean actin bundles. Movement of pollen tube organelles proceeded from the pointed end to the barbed end of the actin filaments of the characean cells. The reconstituted movement was not inhibited by vanadate. KCL at higher concentrations inhibited the movement. Furthermore, heavy meromyosin (HMM) prepared from rabbit skeletal muscle myosin partially inhibited the reconstituted movement and pCMB-modified HMM inhibited it completely. The present results strongly support our previous conclusion that the translocator which generates the motive force of cytoplasmic streaming in pollen tube is myosin.Abbreviations AMP-PNP adenylyl-imidodiphosphate - ATP adenosine-5-triphosphate - ATP--S adenosine-5-0-(3-thiotriphosphate) - BSA bovine serum albumin - CCCP carbonylcyanide m-chlorophenylhydrazone - DTT dithiothreitol - EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis-(-aminoethyl ether)N,N,N,N-tetraacetic acid - HB homogenization buffer - HMM heavy meromyosin - NEM N-ethylmaleimide - pCMB p-chloromercuribenzoic acid - PIPES piperazine-N,N-bis-(2-ethanesulfonic acid) - PPi pyrophosphate     

Pollen-pistil size correlation and pollen size-number trade-off in species of Argentinian Nyctaginaceae with different pollen reserves

Data on pollen and pistil traits from 14 Argentinean Nyctaginaceae species with starch or lipids as pollen reserves are presented. We expect differences in the traits between these two groups of species, but the same pattern within each group for (a) the relationship between pollen size and pistil length assuming that pollen tube length is predetermined by provisions in the pollen independently of the pollen reserve type, and for (b) a trade-off between size and number because available resources for male function are not unlimited. In particular we expect that (a) species with longer pistils will have larger pollen grains, (b) pollen grain size and the number of pollen grains per flower will be negatively correlated. Significant differences in the mean pollen number per flower and mean pollen size were observed between species with different pollen reserve type but not for pistil length. Then, correlation analyses were performed for species with starchy pollen or with pollen with lipids separately. Pollen size - pistil length correlation was positive and significant for species with starchy pollen but not for species with pollen with lipids. On the other hand, pollen size - number correlation was not significant for both starchy and oil-rich species. Results suggest that pollen reserve type would be a relevant factor that constraint pollen size in species of Nyctaginaceae, and that this pollen trait should be considered when studying pollen-pistil relationships, mainly between species of those families with mixed pollen reserves.     

Pollen viability and pollen vigor

Summary Investigations were carried out to correlate pollen viability, assessed on the basis of a fluorochromatic reaction (FCR) test, with pollen vigor, assessed on the basis of the time taken for in vitro germination in pollen grains subjected to high humidity (>95% RH) and temperature (38 °C) or storage stress of Nicotiana tabacum, Agave sp., Tradescantia virginiana, and Iris sp. Both high RH and temperature, as well as storage stresses, affected pollen vigor before affecting pollen viability. The results are discussed in the light of available data on the viability and vigor of stressed pollen and of aged seeds. The need for consideration of pollen vigor, particularly in stored pollen, the inadequacy of the methods presently used, and some of the methods suitable to assess pollen vigor are elaborated.     

Pollen carbohydrates and water content during development, presentation, and dispersal: a short review

Summary. Pollen accumulates starch reserves during development and the final stage of ripening. Before the anther opens, starch is totally or partially converted to pectins, glucose, fructose, sucrose, and to some unknown polysaccharides. Pollen is exposed to dispersing agents in an arrested developmental state which differs according to pollen water content. Pollen is classified as partially dehydrated or partially hydrated. The final water content may be reached before or after anther opening. Especially during exposure and dispersal, partially dehydrated pollen may interconvert soluble and insoluble reserves, modifying internal turgor pressure and hindering water loss or gain. Partially hydrated pollen is commonly devoid of mechanisms to conserve viability in time but has the advantage of quickly emitting pollen tubes on reaching the stigma. Correspondence and reprints: Department of Environmental Sciences, Siena University, 4 Via P.A. Mattioli, 53100 Siena, Italy.     

In vitro chemotropism of pearl millet pollen tubes to stigma tissue: a response to glucose produced in the medium by tissue-bound invertase

Summary Water-homogenized stigma pellets of pearl millet and precipitates resulting from dialysis of their salt extracts were observed to: (1) chemotropically attract pearl millet pollen tubes on a sucrose-containing pollen germination and growth medium, (2) have acid invertase activity as assayed by the arsenomolybdate method, (3) hydrolyze sucrose in the pollen germination and growth medium to glucose as assayed by coupled glucose oxidation with Nitro Blue Tetrazolium, and (4) lose chemotropic and invertase activities upon heat treatment. The results indicate that the in vitro chemotropic attraction of pearl millet pollen tubes to water-homogenized stigma pellets is a response to glucose produced by homogenate-pellet-bound invertase hydrolyzing the sucrose present in the pollen germination and growth medium. Yeast and tomato invertases used as controls verified this conclusion. Water extracts of whole stigmas contained water-soluble acid invertase. The results are discussed in relation to the identification of possible in vivo chemotropic factors of pearl millet and other plants by in vitro assays.Abbreviations dH₂O Deionized, house-distilled water - NBT Nitro Blue Tetrazolium, NBT-medium - PGG medium, pollen germination and growth medium (10% sucrose, 1 mM H₃BO₃, and 1% agarose); - WHS pellet, water-homogenized stigma pellet On Specific Cooperative Agreement 58-6612-8-002 with the Department of Biochemistry, University of Georgia, Athens, GA 30602, USA     

Delay of early fruitlet abscission by branch girdling in citrus coincides with previous increases in carbohydrate and gibberellin concentrations

Current evidence in citrus indicates that gibberellins (GAs) are main determinants of early fruit set while subsequent growth of developing fruits is mostly dependent upon carbohydrate availability. In this work, branch girdling performed at anthesis in Satsuma mandarin (Citrus unshiu (Mak.) Marc.) cv. Okitsu transitorily reduced early abscission rates (12–32 days after anthesis, DAA) delaying initially the process of natural fruitlet drop. The effects of girdling on growth, gibberellin (GA) and carbohydrate concentrations in developing ovaries and fruitlets were assessed during this initial growth stage (0–69 DAA). In girdled branches, abscission rate reduction was preceded by elevated concentrations of carbohydrate and GA in developing ovaries and fruitlets. Girdling at anthesis stimulated higher hexose (21 DAA) and starch (6–20 DAA) concentrations and also higher GA₁ (6 DAA), GA₁₉ (13–20 DAA) and GA₂₀ (6–20 DAA). The results established a relationship between the reduction of early abscission rates and higher concentrations of carbohydrates and GAs induced by girdling in developing fruitlets. These findings revealed that girdling certainly increased GA concentration and strongly suggested that its effect on early fruitlet abscission delay is likely mediated by both GA and carbohydrates.     

Metabolism of carbohydrate and lipid reserves in germinated cotton seeds

Utilization of reserve lipid and carbohydrates during germination (0–12 h) and postgerminative growth (12–48 h) was studied in cotton (Gossypium hirsutum L.) seedlings. Raffinose and stachyose were utilized during the germination period and early growth; mobilization was associated with -galactosidase (EC 3.2.1.22) activity. Results from pulse-chase experiments with [³H]raffinose supplied exogenously to 4-h soaked seeds indicated that raffinose-derived catabolites contributed to the coincident increase in cotyledon sucrose and starch, and to the small increase in axis dry weight. Starch appears to be an alternative sink for end products of hydrolysis of reserve carbohydrates prior to the onset of rapid axis growth and cotyledon expansion. Mobilization of neutral lipid commenced at about 16 h after soaking, concomitant with development of key glyoxylate-cycle and other gluconeogenesis-related enzyme activities. Axis dry weight increased three-fold between 24 and 48 h. Results from pulse-chase (3 h, 16 h) experiments in which [2-¹⁴C]acetate was supplied to cotyledons of intact 22-h-old seedlings showed that acetate-derived metabolites were not transported exclusively to the axes, but were partitioned between axes and cotyledons. Only 27% of total incorporated radioactivity was recovered in axes following the chase, 18% was evolved as CO₂, and the rest was recovered in water-soluble substances (20%) and polymers (31%) within the cotyledons. Of the polymers, 55% of the activity was in polysaccharides (Starch, pectic substances, hemicellulose, cellulose), 25% in protein, and 20% in unidentified neutral and acidic compounds. Considering these data, the amount of lipid mobilized, and various routes by which supplied [2-¹⁴C]acetate could be metabolized, it appears that lipidderived compounds contribute only 25–40% of axis dry-weight gain. Lipid-derived substances retained in the cotyledons likely are utilized for expansion and differentiation of the cotyledons into photosynthetic organs.     

Classification and counting of fluorescent pollen using an image analysis system

Pollen viability is commonly assessed by fluorochromatic reaction (FCR) because of the high correlation between positive fluorescence of the pollen grains and their ability to germinate. One of the advantages of this method is its simplicity. An experiment to test FCR analysis for reproducibility, however, showed that results are affected by subjectivity. There is little consistency between analysts, and assessment by the same analyst may differ for the same pollen sample image examined at different times. These problems were solved by a computerized image analysis system that provides a method for classifying positive and negative fluorescent pollen and automatic counting of the grains in each class. The computerized image analysis system does not change the biochemistry of the FCR test, but avoids some experimental errors owing to the subjectivity of the analyst. Microscope images of the pollen after FCR were digitized and later analyzed by specially designed software, "Plant Meter." This software deletes the dark background of the image to isolate the grains, and subsequently counts positive and negative fluorescent pollen grains. An experiment was carried out to validate software output and it showed reliable results. Moreover, the software is user friendly and very little training is necessary for analysts to achieve reliable results.     

Drought-induced male sterility in rice: Changes in carbohydrate levels and enzyme activities associated with the inhibition of starch accumulation in pollen

Male reproductive development of rice (Oryza sativa L.) is very sensitive to drought. A brief, transitory episode of water stress during meiosis in pollen mother cells of rice grown under controlled environmental conditions induced pollen sterility. Anthers containing sterile pollen were smaller, thinner, and often deformed compared to normal anthers of well-watered plants. Only about 20% of the fully developed florets in stressed plants produced grains, compared to 90% in well-watered controls. Water stress treatments after meiosis were progressively less damaging. Levels of starch and sugars and activities of key enzymes involved in sucrose cleavage and starch synthesis were analyzed in anthers collected at various developmental stages from plants briefly stressed during meiosis and then re-watered. Normal starch accumulation during pollen development was strongly inhibited in stress-affected anthers. During the period of stress, both reducing and non-reducing sugars accumulated in anthers. After the relief of stress, reducing sugar levels fell somewhat below those in controls, but levels of non-reducing sugars remained higher than in controls. Activities of acid invertase and soluble starch synthase in stressed anthers were lower than in controls at comparable stages throughout development, during as well as after stress. Stress had no immediate effect on ADP-glucose pyrophosphorylase activity, but had an inhibitory aftereffect throughout post-stress development. Sucrose synthase activity, which was, relatively speaking, much lower than acid invertase activity, was only slightly suppressed by stress. The results show that it is unlikely that pollen sterility, or the attendant inhibition of starch accumulation, in water-stressed rice plants are caused by carbohydrate starvation per se. Instead, an impairment of enzymes of sugar metabolism and starch synthesis may be among the potential causes of this failure.     

Locomotion and the pollen hoarding behavioral syndrome of the honeybee (<Emphasis Type="Italic">Apis mellifera</Emphasis> L.)

Honeybees selected for the colony level phenotype of storing large quantities of pollen (pollen hoarding) in the nest exhibit greater walking activity than those selected against pollen hoarding. In this study, we use a simple walking assay to demonstrate that walking activity increases with the proportion of high pollen-hoarding alleles in pure and backcrossed strains of bees (high-strain bees > offspring generated from a high backcross > offspring generated from a low backcross > low-strain bees). The trait is heritable but is not associated with markers linked to three quantitative trait loci (QTL) mapped for their effects on pollen hoarding with demonstrated pleiotropic effects on pollen and nectar foraging and learning behavior. However, locomotion in non-selected bees is correlated with responsiveness to sucrose, a trait that correlates with foraging and learning behavior. We propose that pollen-hoarding behavior involves a syndrome of behavioral traits with complex genetic and regulatory architectures that span sensory sensitivity, foraging behavior, and learning. We propose that locomotor activity is the component of this syndrome and reflects the early maturation of the bees that become pollen foragers.     

Long-term storage of Pennisetum glaucum (L.) R. Br. pollen

Summary Pearl millet [Pennisetum glaucum (L.) R. Br.] pollen has been successfully stored for 2,615 and 2,911 days at -18° and -73 °C, respectively, and continues to be viable. Viability of pollen stored at -73 °C appears to be little affected either by pollen storage moisture contents below 7.2% or by storage in glass vial or zip-lock plastic bag containers. Pollen moisture content appears to be more critical for maintaining viability at -18°C than at -73°C. Glass vials appear to be more desirable for longer term (>3 years) storage at -18°C.     

Changes in lipids and polysaccharides during pollen ontogeny in Oenothera anthers

Summary Sudan B and PAS reactions were used for the histochemical detection of lipids and polysaccharides with light microscopy in semithin slides of resin-embedded Oenothera hookeri De Vries anthers at different ontogenic stages. The aim was to establish the variations that occur in both kinds of metabolic products during normal pollen development.In reproductive cells a wave of amylolysis/amylogenesis is observed simultaneously with a lipidic wave. Starch in diploid PMCs is different from starch in the haploid vegetative cell. The gametogenic cell produces neither reserve lipids nor starch. Callose is seen as brilliant white with Sudan B, but is PAS negative at the PMC stage, becoming PAS positive at the tetrad stage. The exine has interbedded lipopolysaccharidic materials. Tapetal plastids show an intense production of lipidic bodies after the vacuolated microspore stage. Lipidic pollen-kitt is present in the immature pollen grains and becomes dissolved at anthesis. Tapetal cells have no cellulosic cell walls from the PMC stage onwards, but they do have a glycocalix. These events have evolutionary significance and will be compared with male-sterile development.Grant of the CONICET-PID 3014000/88 and of the CONICOR-Sub. number 1574/90

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Seasonal distribution of pollen in the atmosphere of melbourne: an airborne pollen calendar

Environmental monitoring of pollen grains in the atmosphere of Melbourne has been achieved using Burkard volumetric traps. Twenty-two families of flowering plants and confiers were identified in the pollen counts. About 62% of these pollen grains belonged to trees, 20% to grasses and 9% to herbs and weedy plants. During spring and summer, the atmosphere contained about 70% of the total annual pollen count. Tree pollen, predominantly elm and cypress, occurred abundantly in late winter and spring, with grass pollen predominantly in spring and early summer. These three types of pollen grains occurred in significant amounts, together accounting for more than 60% of the total annual catch. A seasonal incidence chart (pollen calendar) for Melbourne based on 2 years observation has been constructed. This pollen calendar is useful in identifying sources of allergies against particular seasonal airborne pollen types. Comparison of the time of occurrence of a particular pollen type using the pollen calendar and the time of allergic symptoms, can lead to accurate diagnosis and preventive measures being taken. This study has confirmed that grass pollen is the major source of allergenic pollen in the external environment triggering hay fever and allergic asthma in spring and early summer in Melbourne, Australia.     

Localisation of a carbohydrate epitope recognised by human IgE in pollen of Cupressaceae

The objectives of the present study were: (1) to localise, at the subcellular level, the allergens in pollen of Cupressaceae species, using a monoclonal antibody (mAb 5E6) that is specific for carbohydrate epitopes of allergenic components of Cupressus arizonica pollen extract; (2) to determine whether the glycidic epitope recognised by mAb 5E6 was present in pollen of allergenic species taxonomically unrelated to Cupressaceae; and (3) to determine whether human IgE purified from monosensitive patients recognises the same epitope as mAb 5E6 in Cupressaceae pollen. Immunogold labelling of mAb 5E6 showed a high density of gold particles on the orbicules, supporting the hypothesis that they are important vectors of allergens. A high density was also found on the exine and in the cytoplasm, with the latter finding confirming that fragments of pollen ruptured under humid conditions can represent a vector. The glycidic epitope recognised by mAb 5E6 was detected in all of the species taxonomically unrelated to Cupressaceae, although with varying density. Human IgE recognised the same epitope as mAb 5E6. These findings are consistent with observations of diffuse allergenic cross-reactivity among various allergens. The in situ localisation of a common epitope recognised by both a monoclonal antibody and human IgE could be of importance in immunotherapy.