Proteomic analyses of apoplastic proteins from germinating Arabidopsis thaliana pollen

Pollen grains play important roles in the reproductive processes of flowering plants. The roles of apoplastic proteins in pollen germination and in pollen tube growth are comparatively less well understood. To investigate the functions of apoplastic proteins in pollen germination, the global apoplastic proteins of mature and germinated Arabidopsis thaliana pollen grains were prepared for differential analyses by using 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE) saturation labeling techniques. One hundred and three proteins differentially expressed (p value≤0.01) in pollen germinated for 6h compared with un-germination mature pollen, and 98 spots, which represented 71 proteins, were identified by LC-MS/MS. By bioinformatics analysis, 50 proteins were identified as secreted proteins. These proteins were mainly involved in cell wall modification and remodeling, protein metabolism and signal transduction. Three of the differentially expressed proteins were randomly selected to determine their subcellular localizations by transiently expressing YFP fusion proteins. The results of subcellular localization were identical with the bioinformatics prediction. Based on these data, we proposed a model for apoplastic proteins functioning in pollen germination and pollen tube growth. These results will lead to a better understanding of the mechanisms of pollen germination and pollen tube growth.     

Proteomic identification of differentially expressed proteins in mature and germinated maize pollen

The identification of proteins involved in pollen germination and tube growth is important for fundamental studies of fertility and reproduction in flowering plants. We used 2-DE and MALDI-TOF-MS to identify differentially expressed proteins in mature (P0) and 1-h germinated (P1) maize pollen. Among about 470 proteins separated in 2D gels, the abundances of 26 protein spots changed (up- or down-regulation) between P0 and P1. The 13 up-regulated protein spots were mainly involved in tube wall modification, actin cytoskeleton organization, energy metabolism, signaling, protein folding and degradation. In particular, pectin methylesterase, inorganic pyrophosphatase, glucose-1-phosphate uridylyltransferase and rab GDP dissociation inhibitor α are highly up-regulated, suggesting their potential role in pollen tube growth. The down-regulated 13 protein spots mainly include defense-related proteins, pollen allergens and some metabolic enzymes. This study would contribute to the understanding of the changes in protein expression associated with pollen tube development.     

Characterization of a gene family abundantly expressed in Oenothera organensis pollen that shows sequence similarity to polygalacturonase.

We have isolated and characterized cDNA clones of a gene family (P2) expressed in Oenothera organensis pollen. This family contains approximately six to eight family members and is expressed at high levels only in pollen. The predicted protein sequence from a near full-length cDNA clone shows that the protein products of these genes are at least 38,000 daltons. We identified the protein encoded by one of the cDNAs in this family by using antibodies to beta-galactosidase/pollen cDNA fusion proteins. Immunoblot analysis using these antibodies identifies a family of proteins of approximately 40 kilodaltons that is present in mature pollen, indicating that these mRNAs are not stored solely for translation after pollen germination. These proteins accumulate late in pollen development and are not detectable in other parts of the plant. Although not present in unpollinated or self-pollinated styles, the 40-kilodalton to 45-kilodalton antigens are detectable in extracts from cross-pollinated styles, suggesting that the proteins are present in pollen tubes growing through the style during pollination. The proteins are also present in pollen tubes growing in vitro. Both nucleotide and amino acid sequences are similar to the published sequences for cDNAs encoding the enzyme polygalacturonase, which suggests that the P2 gene family may function in depolymerizing pectin during pollen development, germination, and tube growth. Cross-hybridizing RNAs and immunoreactive proteins were detected in pollen from a wide variety of plant species, which indicates that the P2 family of polygalacturonase-like genes are conserved and may be expressed in the pollen from many angiosperms.     

Inhibition of RNA and protein synthesis in pollen tube development of Pinus bungeana by actinomycin D and cycloheximide

* The effects of actinomycin D and cycloheximide on RNA and protein synthesis were investigated during pollen tube development of Pinus bungeana. * RNA and protein contents, protein expression patterns, cell wall components and ultrastructural changes of pollen tubes were studied using spectrophotometry, SDS-PAGE electrophoresis, Fourier transformed infrared (FTIR) microspectroscopy and transmission electron microscopy (TEM). * Pollen grains germinated in the presence of actinomycin D, but tube elongation and RNA synthesis were inhibited. By contrast, cycloheximide inhibited pollen germination and protein synthesis, induced abnormal tube morphology, and retarded the tube growth rate. SDS-PAGE analysis showed that protein expression patterns changed distinctly, with some proteins being specific for each phase. FTIR microspectroscopy established significant changes in the chemical composition of pollen tube walls. TEM analysis revealed the inhibitors caused disintegration of organelles involved in the secretory system. * These results suggested RNA necessary for pollen germination and early tube growth were present already in the pollen grains before germination, while the initiation of germination and the maintenance of pollen tube elongation depended on continuous protein synthesis.     

Lipid synthesis in germinating Tradescantia pollen

Synthesis of neutral and polar lipids in pollen of Tradescantia paludosa during germination and tube growth was studied by the incorporation of acetate-[1-¹⁴C] into lipids in the presence and absence of inhibitors of RNA and protein synthesis. The proteins required for the synthesis of both neutral lipids and phospholipids are not made de novo during germination but are already present in the mature ungerminated pollen grain and they are functionally stable during the first 2 hr of pollen growth.     

Changing patterns of nucleic acids, basic and acidic proteins in generative nuclei during pollen germination and pollen tube growth in Hippeastrum belladona

Generative and vegetative nuclei of mature and germinated pollen grains from Hippeastrum belladonna were separated in a continuous Ficoll gradient. Less than 3% contamination was observed between the generative and vegetative nuclear fractions. The vegetative nuclei were composed of two populations; the larger population consisted of nuclei with 1C levels of DNA and the smaller with 2C levels. The generative nuclei consisted of a homogeneous population composed of nuclei possessing 2C levels of DNA. Histone synthesis did not occur in vegetative nuclei. Changes appeared in the gel-electrophoretic banding patterns of the F1 histones of vegetative nuclei during germination. Changes were not observed in the generative nuclei. A reduction of general proteins and RNA was observed in vegetative nuclei by 20 h of germination. The phenol-soluble nuclear proteins of vegetative nuclei revealed transitions in electrophoretic banding patterns during pollen germination that were greater than those shown by the histones. These changes in the PSNP primarily involved reduced concentrations of certain proteins rather than synthesis of new ones. However, a new band was observed in the electrophoretic pattern of the PSNP of vegetative nuclei after 12 h of pollen tube growth. No transition was seen in the PSNP of generative nuclei during pollen germination and tube growth. The regulatory role of the PSNP in cell differentiation is discussed in the light of these findings.     

山玉兰花粉离体萌发和花粉管生长特性的研究

山玉兰(Magnolia delavayi)是木兰科木兰属的常绿乔木或大型灌木,是重要的园林造景、庭院绿化素材,也是重要的育种资源。山玉兰花粉的研究对其杂交育种的成败具有重要影响,但目前尚未见其花粉活力的相关报道。该研究以新鲜的山玉兰花粉为对象,采用悬滴培养法分析了温度、光照以及培养液的蔗糖和硼酸浓度对山玉兰花粉萌发的影响。结果表明:(1)山玉兰花粉萌发时,最适宜的温度为27℃。(2)光暗条件下,山玉兰花粉以浓度为5%的蔗糖培养效果最佳,其萌发率在16%以上;而硼酸浓度则以0.001%的培养效果最佳。(3)蔗糖与硼酸共同作用可有效促进花粉萌发和花粉管生长。其中,在光照条件下,以5.0%蔗糖+0.001%硼酸为最适宜的培养液,花粉萌发率达41.27%,花粉管长达281.49μm;而在黑暗条件下,则以5.0%蔗糖+0.01%硼酸为最适宜的培养液,花粉萌发率达45.71%,花粉管长达254.00μm。该研究结果为进一步开展人工辅助授粉、发掘山玉兰的种质资源工作奠定了基础。     

Molecular and physiological characterisation of a 14-3-3 protein from lily pollen grains regulating the activity of the plasma membrane H+ ATPase during pollen grain germination and tube growth

A 14-3-3 protein has been cloned and sequenced from a cDNA library constructed from mRNAs of mature pollen grains of Lilium longiflorum Thunb. Monoclonal antibodies (MUP 5 or MUP 15) highly specific against 14-3-3 proteins recognised a 30-kDa protein in the cytoplasmic fraction of many various lily tissues (leaves, bulbs, stems, anther filaments, pollen grains, stigmas) and in other plants (Arabidopsis seedlings, barley recombinant 14-3-3). In addition, 14-3-3 proteins were detected in a microsomal fraction isolated from pollen grains and tubes, and the amount of membrane-bound 14-3-3 proteins as well as the amount of the plasma membrane (PM) H⁺ ATPase increased during germination of pollen grains and tube growth. No change was observed in the cytoplasmic fraction. A further increase in the amount of 14-3-3 proteins in the microsomal fraction was observed when pollen grains were incubated in germination medium containing 1 μM fusicoccin (FC) whereas the number of 14-3-3s in the cytoplasmic fraction decreased. Fusicoccin also protected membrane-bound 14-3-3 proteins from dissociation after washing with the chaotropic salt KI. Furthermore, FC stimulated the PM H⁺ ATPase activity, the germination frequency and the growth rate of pollen tubes, thus indicating that a modulation of the PM H⁺ ATPase activity by interaction with 14-3-3 proteins may regulate germination and tube growth of lily pollen. Received: 20 June 2000 / Accepted: 2 October 2000     

Effects of high humidity and temperature stress on pollen membrane integrity and pollen vigour in Nicotiana tabacum

Summary The prolonged exposure of pollen Nicotiana tabacum to high humidity at both room temperature and 38° C did not affect membrane integrity as revealed by the fluorochromatic reaction (FCR) test, but did affect pollen vigour. At room temperature germination was not affected, although tube growth was reduced; at 38° C, there was both a reduction in tube growth and delayed germination. When the pollen was subjected to 1 h hydration followed by 1 h desiccation (up to a maximum of four cycles) at room temperature, a reduction in the FCR, germination and tube length after each desiccation treatment was observed. Subsequent hydration fully restored the FCR, but only partially restored germination and tube growth. At 38° C, however, FCR, germination, and tube growth were drastically reduced. The implications of these results on the relationship between FCR and germinability, the responses of pollen exposed to humidity and temperature stress in the field, and on pollen storage are discussed.     

Molecular evidence that most RNAs required for germination and pollen tube growth are stored in the mature pollen grain in petunia

After landing on the stigma, the pollen grain germinates and elongates a tube to deliver its generative nuclei to the egg cell of the ovule. The molecular mechanisms involved in the drastic morphological changes in the pollen grain during this fertilization process remain largely unknown. In this study, the expression of 732 randomly selected genes in petunia pollen and pollen tubes was analyzed by microarray and quantitative PCR analyses. We found no evidence for up-regulation of any of these genes in the pollen tube. Our findings provide support at the gene level for the longstanding hypothesis that pollen germination and tube growth are not dependent on new RNA synthesis and that the large number of RNAs required for germination and tube growth are stored in mature pollen grains.     

In vitro pollen germination and pollen tube growth differences among Quercus robur L. clones in response to meteorological conditions

The impact of meteorological conditions on in vitro pollen germination and pollen tube growth during the initial phases of the development of male flowers in the Pedunculate Oak, Quercus robur, is studied. Phenological observations of male flowers and pollen sampling were performed on the field trial established with grafted Pedunculate Oak clones. During the investigation, weather conditions (absolute minimum and maximum daily air temperature, minimum absolute relative humidity of air and amount of precipitation) were recorded by an automatic meteorological station installed at the field trial. Influence of meteorological conditions on pollen germination and pollen tube growth was studied in the following stages of male flower: (I) during the last ten days of flower bud dormancy, (II) during swelling of the buds, (III) during bud burst and beginning of male catkins elongation, (IV) during the final stage of male flower catkins elongation. High temperatures and low relative air humidity during the bud burst and beginning of the male catkins elongation reduced pollen germination and pollen tube growth. Weather conditions did not significantly affect pollen germination and pollen tube growth during the swelling of flower buds, or in the final stage of male catkins elongation.     

Ectopic expression of Arabidopsis thaliana plasma membrane intrinsic protein 2 aquaporins in lily pollen increases the plasma membrane water permeability of grain but not of tube protoplasts

To investigate the role of aquaporin-mediated water transport during pollen grain germination and tube growth, Arabidopsis thaliana plasma membrane intrinsic proteins (PIPs) were expressed in pollen of Lilium longiflorum (lily). Successful expression of AtPIPs in particle-bombarded lily pollen grains was monitored by co-expression with fluorescent proteins and single-cell RT-PCR, and by measuring the water permeability coefficient (P(os)) in swelling assays using protoplasts prepared from transformed pollen grains and tubes. Expression of AtPIP1;1 and AtPIP1;2 in pollen grains resulted in P(os) values similar to those measured in nontransformed pollen grain protoplasts (6.65 +/- 2.41 microm s(-1)), whereas expression of AtPIP2 significantly increased P(os) (AtPIP2;1, 13.79 +/- 6.38; AtPIP2;2, 10.16 +/- 3.30 microm s(-1)). Transformation with combinations of AtPIP1 and AtPIP2 did not further enhance P(os). Native pollen tube protoplasts showed higher P(os) values (13.23 +/- 4.14 microm s(-1)) than pollen grain protoplasts but expression of AtPIP2;1 (18.85 +/- 7.60 microm s(-1)) did not significantly increase their P(os) values. Expression of none of the tested PIPs had any effect on pollen tube growth rates. The ectopic expression of AtPIP2s in lily pollen increased the water permeability of the plasma membrane in pollen grains, but not in pollen tubes. The measured endogenous water permeability does not limit water uptake during tube growth, but has to be regulated to prevent tube bursting.