In‐Depth Proteome Analysis of Ricinus communis Pollens

Pollen grains are tiny structures vital for sexual reproduction and consequently seed and fruit production in angiosperms, and a source of many allergenic components responsible for deleterious implications for health worldwide. Current pollen research is mainly focused on unraveling the molecular mechanisms underlying the pollen germination and tube formation passing from the quiescent stage. In this context, an in‐depth proteome analysis of the pollens from Ricinus communis at three different stages—that is, mature, hydrated, and in vitro germinated—is performed. This analysis results in the identification of 1950 proteins, including 1773, 1313, and 858, from mature, hydrated, and germinated pollens, respectively. Based on label‐free quantification, 164 proteins are found to be significantly differentially abundant from mature to hydrated pollens, 40 proteins from hydrated to germinated, and 57 proteins from mature to germinated pollens, respectively. Most of the differentially abundant proteins are related to protein, carbohydrate, and energy metabolism and signaling. Besides other functional classes, a reasonable number of the proteins are predicted to be allergenic proteins, previously undiscovered. This is the first in‐deep proteome analysis of the R. communis pollens and, to the best of our knowledge, one of the most complete proteome dataset identified from the pollens of any plant species, thus providing a reference proteome for researchers interested in pollen biology.     

Cloning and characterization of a pollen-specific cDNA encoding a glutamic-acid-rich protein (GARP) from potato Solanum berthaultii

A pollen-specific cDNA was isolated from a cDNA library of in vitro germinated pollen of the diploid potato species Solanum berthaultii. The cDNA clone, designated SB401, hybridizes to a messenger RNA of 1.2 kb length in mature and germinated pollen. SB401 messenger RNA is absent from other parts of the plant, including other flower tissues. SB401 cDNA, which possesses a long stretch of AT-rich 5-untranslated leader sequence, encodes a glutamic acid-rich protein (GARP) which is hydrophilic throughout and contains six imperfect repeated motifs of the sequence V-V-E-K-K-N/E-E with the di-basic amino acid residue pair (K-K) as the core within the repeats. These repeats are spaced at irregular intervals and predicted to form an -helical structure. The SB401 protein was over-expressed in Escherichia coli and the purified protein was used for raising antiserum. Both E. coli-expressed and the endogenous SB401 proteins in pollen and pollen tubes appear much larger on SDS-polyacrylamide gels than their calculated molecular masses. Immunoblotting revealed the protein to be most abundant in germinated pollen. The structural features of SB401 protein and a possible role for the protein in pollen development, pollen germination, and pollen tube growth are discussed.     

Composition of the walls of pollen grains of the seagrass Amphibolis antarctica

The composition of walls isolated from pollen grains of the seagrass Amphibolis antarctica was determined. Glucose, galactose, and rhamnose were the major neutral monosaccharides in the wall polysaccharides, and fucose, arabinose, xylose, and mannose were present in minor proportions. No apiose, a monosaccharide present in the wall polysaccharides of the vegetative parts of the seagrass Heterozostera tasmanica, was found. Large amounts of uronic acid (mainly as galacturonic acid) were found in the walls. The monosaccharides were probably present in cellulose and pectic polysaccharides, the latter comprising neutral pectic galactans, and rhamnogalacturonans containing high proportions of rhamnose. The walls contained a small amount of protein; glycine and lysine were the amino acids present in the highest proportions. Histochemical examination of isolated walls confirmed the presence of polyanionic components (pectic polysaccharides), -glucans (cellulose), and protein. The composition of the walls is discussed in relation to analyses of the walls of pollen grains and vegetative organs of other plants.     

Effect of a myo-Inositol Antagonist, 2-O, C-Methylene-myo-Inositol, on the Metabolism of myo-Inositol-2-H and d-Glucose-1-C in Lilium longiflorum Pollen

2-O,C-Methylene-myo-inositol (MMO), a myo-inositol (MI) antagonist, inhibits germination and tube elongation of pollen from Lilium longiflorum cv. Ace or 44. The presence of 5 mm MMO in Dickinson's pentaerythritol medium (Plant Physiol. 43:1-8) partially blocks germination. The tubes produced are short and fail to elongate. In the presence of MI, MMO's toxic effect is blocked. As little as 0.56 mm MI will maintain normal germination in the presence of 43 mm MMO, and pollen tubes continue to elongate for 2 to 3 hr. Eventually, the toxic action of MMO prevents further growth. MMO does not inhibit UDP-d-glucose dehydrogenase from lily pollen.Uptake of MI-2-(3)H and incorporation of tritium into galacturonic acid and pentose units of tube wall pectin are blocked by MMO. The site of this inhibition is undertermined. Uptake of d-glucose-1-(14)C and incorporation of (14)C into 70% ethyl alcohol-insoluble polysaccharides of germinated pollen are not blocked by MMO, but distribution of label into polysaccharide product is altered. In MMO-treated pollen, very little (14)C is found in uronic acid or pentose units. At 30 mm MMO, about two-thirds of the carbon flow from d-glucose to these pectic components is interrupted. MMO also alters d-glucose metabolism in the 70% ethyl alcohol-soluble fraction, but the compound involved must still be identified.These results offer fresh evidence of an intermediary role for MI during UDP-d-glucuronate biosynthesis in germinated pollen.     

The structure, function, and biosynthesis of plant cell wall pectic polysaccharides

Plant cell walls consist of carbohydrate, protein, and aromatic compounds and are essential to the proper growth and development of plants. The carbohydrate components make up ∼90% of the primary wall, and are critical to wall function. There is a diversity of polysaccharides that make up the wall and that are classified as one of three types: cellulose, hemicellulose, or pectin. The pectins, which are most abundant in the plant primary cell walls and the middle lamellae, are a class of molecules defined by the presence of galacturonic acid. The pectic polysaccharides include the galacturonans (homogalacturonan, substituted galacturonans, and RG-II) and rhamnogalacturonan-I. Galacturonans have a backbone that consists of α-1,4-linked galacturonic acid. The identification of glycosyltransferases involved in pectin synthesis is essential to the study of cell wall function in plant growth and development and for maximizing the value and use of plant polysaccharides in industry and human health. A detailed synopsis of the existing literature on pectin structure, function, and biosynthesis is presented.     

Cell wall reformation by pollen tube protoplasts of olive (Olea europaea L.): structural comparison with the pollen tube wall

. Mature pollen grains of olive (Olea europaea L.) were germinated in vitro in Brewbaker and Kwack medium, and emerging pollen tubes were then enzymatically digested in the presence of high osmoticum. This treatment resulted in simultaneous degradation of pollen tube walls and fragmentation of their cytoplasm, giving rise to numerous protoplasts of different sizes and different numbers of nuclei. After the protoplasts had been purified, they were cultured in Murashige and Skoog medium supplemented with auxin and cytokinin. The initial steps of cell wall reformation were studied after 12 h and 24 h of culture with a series of cytochemical techniques including periodic acid-Schiff reagent and phosphotungstic acid, as well as with electron microscopy and immunocytochemical techniques using monoclonal antibodies directed against pectins and #-(1̅)-glucan (callose). Among the components of new wall in the protoplasts, callose proved to be the earliest and most abundantly secreted polysaccharide, whereas the deposition of pectins recognized by the antibody JIM7 started several hours later. Pectins that bind JIM5 antibody were not detected in this early stage of development. Cell wall components deposited by protoplasts were compared with those present in growing pollen tubes. Callose secreted by protoplasts formed a relatively thicker layer than that found in the tubes, and pectins recognized by JIM7 were highly abundant, mostly within the cytoplasm and in the apical zone of the tubes.     

Ferulic acid is esterified to glucuronoarabinoxylans in pineapple cell walls

The ester-linkage of ferulic acid (mainly E) to polysaccharides in primary cell walls of pineapple fruit (Ananas comosus) (Bromeliaceae) was investigated by treating a cell-wall preparation with 'Driselase' which contains a mixture of endo- and exo-glycanases, but no hydroxycinnamoyl esterase activity. The most abundant feruloyl oligosaccharide released was O-[5-O-(E-feruloyl)-alpha-L-arabinofuranosyl](1-->3)-O-beta-D-xylopyranosyl-(1-->4)-D-xylopyranose (FAXX). This indicated that the ferulic acid is ester-linked to glucuronoarabinoxylans in the same way as in the primary walls of grasses and cereals (Poaceae). Glucuronoarabinoxylans are the major non-cellulosic polysaccharides in the pineapple cell walls.     

The block of intracellular calcium release affects the pollen tube development of Picea wilsonii by changing the deposition of cell wall components

Two potent drugs, neomycin and TMB-8, which can block intracellular calcium release, were used to investigate their influence on pollen tube growth and cell wall deposition in Picea wilsonii. Apart from inhibiting pollen germination and pollen tube growth, the two drugs largely influenced tube morphology. The drugs not only obviously disturbed the generation and maintenance of the tip-localized Ca(2+) gradient but also led to a heavy accumulation of callose at the tip region of P. wilsonii pollen tubes. Fourier transform infrared (FTIR) spectroscopy analysis showed that the deposition of cell wall components, such as carboxylic acid, pectins, and other polysaccharides, in pollen tubes was changed by the two drugs. The results obtained from immunolabeling with different pectin and arabinogalactan protein antibodies agreed well with the FTIR results and further demonstrated that the generation and maintenance of the gradient of cross-linked pectins, as well as the proportional distribution of arabinogalactan proteins in tube cell walls, are essential for pollen tube growth. These results strongly suggest that intracellular calcium release mediates the processes of pollen germination and pollen tube growth in P. wilsonii and its inhibition can lead to abnormal growth by disturbing the deposition of cell wall components in pollen tube tips.     

Dynamic of polysaccharide and lipid in the developing microsporangiums of Chinese fir (Cunninghamia lanceolata)

Microsporongium development in Chinese fir (Cunninghamia lanceolata) was investigated using cytochemical methods with a special attention to the fluctuations (in amount and distribution) of polysaccharide and lipid reserves along the development of the microsporangium. Semi-thin sections of microsporangia at different developmental stages were stained with periodic acid–Schiff (PAS) reagent and Sudan Black B to detect insoluble polysaccharides and neutral lipids, respectively. In young microsporangia, microspore mother cells began to accumulate starch grains and lipids, which disappeared during microspore development. Following microspore division, the starch grains present in bicellular pollen disappeared and abundant lipid deposits were accumulated. In mature pollen, only abundant lipids accumulated as storage material. The pollen wall of C. lanceolata is predominantly composed of polysaccharidic intine, and the sporopollenin-containing exine is weakly developed and only forms a thin layer covering the intine.     

A histochemical study of meiocytes, microspores, pollen and the tapetum in Kalanchoe

A histochemical study was made of developing sporogenous cells, meiocytes, microspores, pollen and the tapetum in anthers of Kalanchoë morlagei. Storage polysaccharides were seen only in mature pollen. Ascorbic acid was not found in the sporogenous cells, but in meiocytes a high quantity of this compound occurred in the cytoplasm. Spore tetrads, microspores and pollen also had a high ascorbic acid content. The amounts of RNA and proteins were high in the sporogenous cells and in meiocytes during meiosis–I, but a small reduction trend with respect to RNA content was noticed. Microspores in the tetrad showed high amounts of RNA and proteins. In the young microspores RNA and proteins declined. Later, as the microspores matured, an increase in content of RNA and proteins took place. The wall of the young microspores gave a faint green colour with azure B stain, the intensity of which increased and remained high in the exine of the mature pollen. The additional wall thickening around the meiocytes and tetrads gave a strong pink colour with PAS test. This thickening showed presence of silver granules when tested for ascorbic acid, the tapetum synthesized abundant quantities of PAS positive starch, ascorbic acid, RNA and proteins from its appearance in the anther wall until microspore formation. During meiocyte meiosis the tapetum became highly vesicular. Our results indicate that the tapetum constitutes a tissue specialized for storing and supplying basic nutritive substances for the developing pollen in the anther.     

Protein synthesis in tobacco pollen tubes: preferential synthesis of cell-wall 69-kDa and 66-kDa glycoproteins

One- and two-dimensional electrophoresis of Nicotiana tabacum pollen and pollen tube proteins confirmed that a new protein is preferentially synthesized during pollen germination and tube growth and becomes the most abundant protein in pollen tubes. Analysis of proteins extracted with sodium dodecyl sulfate (SDS) from different pollen tube fractions showed that it is the most abundant non-covalently bound wall protein, characterized by molecular mass of 69 kDa, pI between 7.9 and 8.2, and glycosylation with glucose and/or mannose. Amino acid analysis revealed relative abundance of serine, glutamic acid and glycine, but did not show the presence of hydroxyproline. According to all these characteristics, it cannot be classified as an extensin-like protein. Another prominent wall-bound glycoprotein has a molecular mass of 66 kDa and the same pI as the 69 kDa glycoprotein. These two glycoproteins are similar also in ConA binding, rate of synthesis, and rapid incorporation into pollen tube walls. Their synthesis is strongly reduced by tunicamycin and this inhibition results in the occurrence of new polypeptides in the range of 57–61 kDa. Tunicamycin also inhibited pollen tube growth. At 10 ng ml^-1 and 50 ng ml^-1 the inhibitor reduced pollen tube mass after 24 h of culture by 30% and 85%, respectively. This indicates that tobacco pollen presents a system highly sensitive to tunicamycin and that cotranslational N-linked glycosylation on the rough endoplasmic reticulum is required for 66 and 69 kDa glycoprotein formation and for pollen tube growth. Although other proteins appear during pollen germination and tube growth, the new proteins occur at low levels and seem to originate through modifications of preexisting polypeptides. In contrast to 69 and 66 kDa proteins, most proteins detected by [¹⁴C]amino acid incorporation and fluorography of gels were not revealed by Coomassie blue staining.     

Activity of cell-wall degradation associated with differentiation of isolated mesophyll cells of Zinnia elegans into tracheary elements

Cell walls were prepared from cultured mesophyll cells of Zinnia elegans L. that were transdifferentiating into tracheary elements and incubated in a buffer to undergo autolysis. The rate of autolysis of cell walls was determined by measuring the amount of carbohydrate released from the cell walls into the buffer during incubation. During the course of culture of mesophyll cells, the autolysis rate increased markedly at the time when thickenings of secondary cell walls characteristic of tracheary elements became visible (after 48-72 h of culture), and thereafter the rate remained at a high level. Comparative studies on the autolysis rate of cell walls using various control cultures, in which tracheary element differentiation did not take place, revealed a close relationship between the autolysis rate around the 60th hour of culture and differentiation. Sugar analysis by colorimetric assays and gas chromatography of carbohydrates released from the cell walls detected uronic acid, arabinose, galactose, glucose, xylose, rhamnose, fucose, and mannose. Among these sugars, uronic acid was the most abundant, and accounted for approximately half of the total released sugars. The decrease of acidic polysaccharides in the primary cell walls during tracheary element differentiation was visualized by staining cultured cells with alcian blue at pH 2.5. These results suggest that active degradation of components of primary cell walls, including pectin, is integrated into the program of tracheary element differentiation.     

Expression patterns of conserved microRNAs in the male gametophyte of loblolly pine (Pinus taeda)

MicroRNAs (miRNAs) are small RNAs that regulate genes involved in various aspects of plant development, but their presence and expression patterns in the male gametophytes of gymnosperms have not yet been established. Therefore, this study identified and compared the expression patterns of conserved miRNAs from two stages of the male gametophyte of loblolly pine (Pinus taeda), which are the mature (ungerminated) and germinated pollen. Microarray was used to identify conserved miRNAs that varied in expression between these two stages of the loblolly pine male gametophyte. Forty-seven conserved miRNAs showed significantly different expression levels between mature and germinated loblolly pine pollen. In particular, miRNAs representing 14 and 8 families were up- and down-regulated in germinated loblolly pine pollen, respectively. qRT-PCR was used to validate their expression patterns using representative miRNAs. Target genes and proteins were identified using psRNATarget program. Predicted targets of the 22 miRNA families belong mostly to classes of genes involved in defense/stress response, metabolism, regulation, and signaling. qRT-PCR was also used to validate the expression patterns of representative target genes. This study shows that conserved miRNAs are expressed in mature and germinated loblolly pine pollen. Many of these miRNAs are differentially expressed, which indicates that the two stages of the male gametophyte examined are regulated at the miRNA level. This study also expands our knowledge of the male gametophytes of seed plants by providing insights on some similarities and differences in the types and expression patterns of conserved miRNAs between loblolly pine with those of rice and Arabidopsis.     

LeProT1, a transporter for proline, glycine betaine, and gamma-amino butyric acid in tomato pollen

During maturation, pollen undergoes a period of dehydration accompanied by the accumulation of compatible solutes. Solute import across the pollen plasma membrane, which occurs via proteinaceous transporters, is required to support pollen development and also for subsequent germination and pollen tube growth. Analysis of the free amino acid composition of various tissues in tomato revealed that the proline content in flowers was 60 times higher than in any other organ analyzed. Within the floral organs, proline was confined predominantly to pollen, where it represented >70% of total free amino acids. Uptake experiments demonstrated that mature as well as germinated pollen rapidly take up proline. To identify proline transporters in tomato pollen, we isolated genes homologous to Arabidopsis proline transporters. LeProT1 was specifically expressed both in mature and germinating pollen, as demonstrated by RNA in situ hybridization. Expression in a yeast mutant demonstrated that LeProT1 transports proline and gamma-amino butyric acid with low affinity and glycine betaine with high affinity. Direct uptake and competition studies demonstrate that LeProT1 constitutes a general transporter for compatible solutes.     

Purification of maize pollen exines and analysis of associated proteins

Zea mays (maize) pollen exines have been purified with the use of differential centrifugation and sucrose gradients, followed by mild detergent and high salt treatment. The final exine fraction is highly purified from other organelles and subcellular structures as assayed by transmission electron microscopy. Using mature maize pollen as the starting material, 0.2 to 0.3% of the total pollen protein remained associated with the exine fraction throughout the purification. Seven abundant sodium dodecyl sulfate-extractable proteins are detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the final fraction. Amino acid analysis reveals that one of the proteins contains a substantial amount of hydroxyproline, a characteristic of some primary cell wall proteins. The amino acid composition of the 25-kD protein strongly implies that it is an arabinogalactan protein. When exines are purified from earlier pollen developmental stages, a subset of the proteins found in the mature pollen exine is seen.     

An electron microscopic study of the mature megagametophyte in Zea mays

With light microscopy maize megagametophytes stained with Alcian blue-periodic acid-Schiff (AB-PAS) reveal acid or neutral polysaccharides in various cell walls. Comparative fine structural studies were made of permanganate- or OsO₄-fixed material. Organelle distribution is random in the vacuolate and multinucleate antipodal cells; organelles are abundant; starch is scarce. Antipodal cell walls have large openings forming several syncytia. Some walls are papillate. In the central cell (primary endosperm cell) a thin peripheral layer of cytoplasm surrounds the large vacuole; organelle number is moderate; starch is abundant. The central cell wall is also papillate adjacent to the antipodals and around the egg apparatus. In the synergids organelle distribution is non-random; nuclei and numerous organelles occupy the micropylar cytoplasm of each synergid; vacuoles dominate the chalazal cytoplasm of these cells. The filiform apparatus stains with AB-PAS and is composed of both lightly and darkly stained amorphous material. In the egg, organelle distribution is perinuclear with vacuoles proximal to the micropyle; mitochondria are large, abundant and polymorphic; starch is abundant. Nucleolar diameter is five times greater in the central cell and egg than in the antipodal cells and ten times greater than in the synergids. Plasmodesmata occur in all cell walls within the gametophyte, but none appear in the gametophyte wall itself. It is suggested that the antipodals and synergids might be secretory, the latter probably being involved in pollen tube attraction, and that stored metabolites in the central cell and egg cytoplasm support rapid increase in metabolism following fertilization.     

Cell-wall polysaccharides and glycoproteins of parenchymatous tissues of runner bean (Phaseolus coccineus).

1. Polymers were solubilized from the cell walls of parenchyma from mature runner-bean pods with minimum degradation by successive extractions with cyclohexane-trans-1,2-diamine-NNN'N'-tetra-acetate (CDTA), Na2CO3 and KOH to leave the alpha-cellulose residue, which contained cross-linked pectic polysaccharides and Hyp-rich glycoproteins. These were solubilized with chlorite/acetic acid and cellulase. The polymers were fractionated by anion-exchange chromatography, and fractions were subjected to methylation analysis. 2. The pectic polysaccharides differed in their ease of extraction, and a small proportion were highly cross-linked. The bulk of the pectic polysaccharides solubilized by CDTA and Na2CO3 were less branched than those solubilized by KOH. There was good evidence that most of the pectic polysaccharides were not degraded during extraction. 3. The protein-containing fractions included Hyp-rich and Hyp-poor glycoproteins associated with easily extractable pectic polysaccharides, Hyp-rich glycoproteins solubilized with 4M-KOH+borate, the bulk of which were not associated with pectic polysaccharides, and highly cross-linked Hyp-rich glycoproteins. 4. Isodityrosine was not detected, suggesting that it does not have a (major) cross-linking role in these walls. Instead, it is suggested that phenolics, presumably linked to C-5 of 3,5-linked Araf residues of Hyp-rich glycoproteins, serve to cross-link some of the polymers. 5. There were two main types of xyloglucan, with different degrees of branching. The bulk of the less branched xyloglucans were solubilized by more-concentrated alkali. The anomeric configurations of the sugars in one of the highly branched xyloglucans were determined by 13C-n.m.r. spectroscopy. 6. The structural features of the cell-wall polymers and complexes are discussed in relation to the structure of the cell walls of parenchyma tissues.