Anna Speranza

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

Abstractu2002Pollen 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.

In vitro germination of apple pollens

Abstract In order to characterize pollen viability and germinability by biochemical parameters, an introductory investigation was made of the germinability in vitro of pollen of 2 apple cultivars, ‘Golden Delicious’ and ‘Starkrimson’, partially self-incompatible and totally self-incompatible, respectively. Best results of percent germination were obtained for both cultivars after 120 min incubation in Petri dishes at 30°C in a medium containing 0.2 M sucrose, 20 μg/ml H 3 BO 3 , 300 μg/ml Ca(NO 3 ) 2 · 4H 2 O. Optimum pH was 6.0 for ‘Starkrimson’ and 7.0 for ‘Golden Delicious’.

Pd-nanoparticles cause increased toxicity to kiwifruit pollen compared to soluble Pd(II).

In the present study, endpoints including in vitro pollen performance (i.e., germination and tube growth) and lethality were used as assessments of nanotoxicity. Pollen was treated with 5-10 nm-sized Pd particles, similar to those released into the environment by catalytic car exhaust converters. Results showed Pd-nanoparticles altered kiwifruit pollen morphology and entered the grains more rapidly and to a greater extent than soluble Pd(II). At particulate Pd concentrations well below those of soluble Pd(II), pollen grains experienced rapid losses in endogenous calcium and pollen plasma membrane damage was induced. This resulted in severe inhibition and subsequent cessation of pollen tube emergence and elongation at particulate Pd concentrations as low as 0.4 mg L(-1). Particulate Pd emissions related to automobile traffic have been increasing and are accumulating in the environment. This could seriously jeopardize in vivo pollen function, with impacts at an ecosystem level.

Reactive oxygen species are involved in pollen tube initiation in kiwifruit.

The role of reactive oxygen species (ROS) during pollen tube growth has been well established, but its involvement in the early germination stage is poorly understood. ROS production has been reported in germinating tobacco pollen, but evidence for a clear correlation between ROS and germination success remains elusive. Here, we show that ROS are involved in germination and pollen tube formation in kiwifruit. Using labelling with dihydrofluorescein diacetate (H(2) FDA) and nitroblue tetrazolium (NBT), endogenous ROS were detected immediately following pollen rehydration and during the lag phase preceding pollen tube emergence. Furthermore, extracellular H(2) O(2) was found to accumulate, beginning a few minutes after pollen suspension in liquid medium. ROS production was essential for kiwifruit pollen performance, since in the presence of compounds acting as superoxide dismutase/catalase mimic (Mn-5,10,15,20-tetrakis(1-methyl-4-pyridyl)21H,23H-porphin, Mn-TMPP) or as NADPH oxidase inhibitor (diphenyleneiodonium chloride, DPI), ROS levels were reduced and pollen tube emergence was severely or completely inhibited. Moreover, ROS production was substantially decreased in the absence of calcium, and by chromium and bisphenol A, which inhibit germination in kiwifruit. Peroxidase activity was cytochemically revealed after rehydration and during germination. In parallel, superoxide dismutase enzymes, particularly the Cu/Zn-dependent subtype - which function as superoxide radical scavengers - were detected by immunoblotting and by an in-gel activity assay in kiwifruit pollen, suggesting that ROS levels may be tightly regulated. Timing of ROS appearance, early localisation at the germination aperture and strict requirement for germination clearly suggest an important role for ROS in pollen grain activation and pollen tube initiation.

The environmental endocrine disruptor, bisphenol A, affects germination, elicits stress response and alters steroid hormone production in kiwifruit pollen

In vitro toxicity of the endocrine disruptor bisphenol A (BPA) to pollen, the male haploid generation of higher plants, was studied. BPA caused significant inhibition of both tube emergence and elongation of kiwifruit pollen in a dose-dependent manner, beginning at 10 mg · l(-1); morphological changes to tubes were also detected. Despite strong inhibition of pollen tube production and growth, a large percentage of treated cells remained viable. Immunoblotting experiments indicated that levels of BiP and 14-3-3, which are proteins involved in stress response, substantially increased in BPA-treated pollen compared to controls. The increases were dose-dependent in the range 10-50 mg · l(-1) BPA, i.e. even when germination ability was completely blocked. Steroid hormones (17 β-estradiol, progesterone and testosterone) were detected in kiwifruit pollen, and their levels increased during germination in basal medium. In a BPA treatment of 30 mg · l(-1), larger increases in both estrogen and testosterone concentrations were detected, in particular, a six-fold increase of 17 β-estradiol over control concentration (30 min). The increased hormone levels were maintained for at least the 90 min incubation. Increasing concentrations of exogenous testosterone and 17 β-estradiol increasingly inhibited pollen tube emergence and elongation. Current data for BPA-exposed kiwifruit pollen suggest a toxicity mechanism that is at least in part based on a dramatic imbalance of steroid hormone production during tube organisation, emergence and elongation. It may be concluded that BPA, a widespread environmental contaminant, can cause serious adverse effects to essential pollen functions. On a broader scale, this chemical poses a potential risk to the reproductive success of higher plants.

Species-dependent chromium accumulation, lipid peroxidation, and glutathione levels in germinating kiwifruit pollen under Cr(III) and Cr(VI) stress.

The accumulation of chromium by germinating kiwifruit pollen appears to be significantly affected by Cr species, Cr concentration and calcium availability. Cr(III) accumulation always occurred in a linear manner while Cr(VI) uptake followed a logarithmic model. In the absence of exogenous calcium, Cr(III) accumulation was much higher than that of Cr(VI). It was observed that, as the Cr(III) concentration increased, there was a significant decrease in the endogenous calcium content of pollen, ultimately leading to complete calcium depletion after 90 min of incubation at 150 microM Cr(III). This loss of calcium could be responsible for the strong inhibition of tube emergence and growth following exposure of pollen to Cr(III). Indeed, when exogenous calcium was added to the kiwifruit pollen culture medium, significant growth recovery and reduced Cr(III) uptake occurred; the opposite was true in Cr(VI)-treatments. A significant rise in lipid peroxide production occurs in the presence of both Cr species; the effect was more pronounced following Cr(VI) exposure. Finally, glutathione pool dynamics appears to be differentially affected by chromium species and concentrations. In conclusion, results of the present study have provided important information regarding the different activity profiles of Cr(III) and Cr(VI) in relation to kiwifruit pollen performance, and have also demonstrated differences in some biochemical responses of pollen to metal stress.

The cell wall of kiwifruit pollen tubes is a target for chromium toxicity: alterations to morphology, callose pattern and arabinogalactan protein distribution

Trivalent chromium has previously been found to effectively inhibit kiwifruit pollen tube emergence and elongation in vitro. In the present study, a photometric measure of increases in tube wall production during germination showed that 25 and 50 mum CrCl(3) treatment induced a substantial reduction in levels of polysaccharides in walls over those in controls. Moreover, chromium-treated kiwifruit pollen tubes had irregular and indented cell walls. Callose, the major tube wall polysaccharide, was deposited in an anomalous punctuate pattern. Arabinogalactan proteins (AGPs), which are integral in maintaining correct tube growth and shape in kiwifruit pollen, were found to be strongly altered in their distribution after CrCl(3) treatment compared to control tube walls. Transmission electron microscopy-immunogold analysis using four monoclonal antibodies (JIM8, JIM13, JIM14 and MAC207) revealed discontinuous AGP distribution within the treated tube walls. Such clearly discernable alterations in the molecular and morphological architecture of pollen tube walls may be detrimental in vivo for the male gametophyte to accomplish its vital role in the fertilisation process.

Insect controlled pollination in Japanese plum (Prunus salicina Lindl.)

Abstract Trials were performed in a commercial orchard of self-incompatible plum, whose pollination and fruit yield often are a real problem in this early blooming crop. For this reason we introduced in the orchard honey bees, mason bees, and bumble bees. The efficiency of pollinator insects was evaluated in the open field by counting—for 3 h a day in five sampling areas of the orchard—the number of visits to the flowers of the main cultivar. No bumble bees were detected visiting the flowers of the main cultivar. The other two pollinators showed a foraging activity which decreases by increasing the distance from their respective starting points. In each sampling area, the total number of visits was strongly related to fruit number and total yield. No significant differences were detected regarding fruit quality. Fruit yield of the most visited areas was very close to that obtained after mechanical pollination by pollen spray. Pollination was also performed on caged trees, using the different insects separately in order to evaluate their respective efficiency.

Biochemical and ultrastructural features related to male sterility in the dioecious species Actinidia deliciosa

Kiwifruit (Actinidia deliciosa var. deliciosa) is a dioecious plant whose flowers are morphologically hermaphrodite yet functionally unisexual. The objective of this study was to investigate the male sterility of female flowers in kiwifruit through an integrated biochemical and cytohistological approach. High levels of free and trichloroacetic acid (TCA)-insoluble conjugated polyamines (PAs) were found to be associated with pollen degeneration. Carbohydrate levels greatly differed when comparing mature to degenerated pollen from the anthers of male-fertile and male-sterile flowers, respectively. In the cytoplasm, carbohydrates strongly decreased in the degenerated pollen, whereas in the cell wall, their levels remained consistent. The degenerated pollen showed a poorly sculptured sexine, an anomalous nexine, and an intine consisting of a single stratum only. Through in vitro culture of anthers from male-sterile plant, the pollen occasionally reached the binucleate stage. The structural anomalies in the pollen of the male-sterile plant and the alterations in PA and carbohydrate contents, together with the results of the in vitro anther culture, suggest that the male sterility in this species could be under both sporophytic and gametophytic control.

In vitro toxicity of silver nanoparticles to kiwifruit pollen exhibits peculiar traits beyond the cause of silver ion release

The vast use of silver nanoparticles (AgNPs) mandates thorough investigation of their impact on biosystems at various levels. The cytotoxicity of PVP coated-AgNPs to pollen, the aploid male gametophyte of higher plants, has been assessed here for the first time. The negative effects of AgNPs include substantial decreases in pollen viability and performance, specific ultrastructural alterations, early changes in calcium content, and unbalance of redox status. Ag⁺ released from AgNPs damaged pollen membranes and inhibited germination to a greater extent than the AgNPs themselves. By contrast, the AgNPs were more potent at disrupting the tube elongation process. ROS deficiency and overproduction were registered in the Ag⁺- and AgNP-treatment, respectively. The peculiar features of AgNP toxicity reflected their specific modes of interaction with pollen surface and membranes, and the dynamic exchange between coating (PVP) and culture medium. In contrast, the effects of Ag⁺ were most likely induced through chemical/physicochemical interactions.