Regulation of Actin Dynamics in Pollen Tubes： Control of Actin Polymer Level

Actin cytoskeleton undergoes rapid reorganization in response to internal and external cues. How the dynamics of actin cytoskeleton are regulated, and how its dynamics relate to its function are fundamental questions in plant cell biology. The pollen tube is a well characterized actin-based cell morphogenesis in plants. One of the striking features of actin cytoskeleton characterized in the pollen tube is its surprisingly low level of actin polymer. This special phenomenon might relate to the function of actin cytoskeleton in pollen tubes. Understanding the molecular mechanism underlying this special phenomenon requires careful analysis of actin-binding proteins that modulate actin dynamics directly. Recent biochemical and biophysical analyses of several highly conserved plant actin-binding proteins reveal unusual and unexpected properties, which emphasizes the importance of carefully analyzing their action mechanism and cellular activity. In this review, we highlight an actin monomer sequestering protein, a barbed end capping protein and an F-actin severing and dynamizing protein in plant. We propose that these proteins function in harmony to regulate actin dynamics and maintain the low level of actin polymer in pollen tubes.     

Subcellular Localization of the S Locus F-box Protein AhSLF-S2 in Pollen and Pollen Tubes of Self-Incompatible Antirrhinum

The distribution of the S locus F-box (SLF) protein was examined by immunocytochemistry and Western blot techniques using an antibody against the C-terminal part of AhSLF-S2 in self-incompatible Iines of Antirrhinum. Abundant gold particles were found where pollen tubes emerge in vitro. With the elongation of pollen tubes, binding sites for the antibody were found in the cytoplasm of the pollen tubes,including the peripheral part of the endoplasmic reticulum. After germination in vitro for 16 h, the product of AhSLF-S2 and possibly its allelic products could still be detectable, implying that the SLF protein has a role in the elongating process of pollen tubes. The present study provides evidence at the protein level that the SLF protein is present in pollen cytoplasm during pollen tube growth. These findings are discussed, as is their potential role in the self-incompatible response in Antirrhinum.     

Golgi 58K-like protein in pollens and pollen tubes ofLilium davidii

In animal cells, Golgi apparatus is located near the microtubule organizing center (MTOC) and its position is determined partly by 58K protein. By sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immuno-blotting methods, a 58K-like protein has been found in pollen grains and pollen tubes of Lilium davidii. Its molecular weight is very similar to that of the 58K protein of animal cells. By immunofluorescence labeling, under a confocal laser scanning microscope (CLSM), the animal 58K antibody revealed a punctate staining in pollen grains and pollen tubes, which is consistent with the distribution of Golgi apparatus in plant cells. In addition, immuno-gold labeling and transmission electron microscopy showed that the 58K-like protein bound mainly to the membrane of vesicles-like structure near Golgi apparatus. This is the first demonstration of the 58K-like protein in plant cells.     

Pollen Tube Growth： a Delicate Equilibrium Between Secretory and Endocytic Pathways

Although pollen tube growth is a prerequisite for higher plant fertilization and seed production, the processes leading to pollen tube emission and elongation are crucial for understanding the basic mechanisms of tip growth. It was generally accepted that pollen tube elongation occurs by accumulation and fusion of Golgi-derived secretory vesicles （SVs） in the apical region, or clear zone, where they were thought to fuse with a restricted area of the apical plasma membrane （PM）, defining the apical growth domain. Fusion of SVs at the tip reverses outside cell wall material and provides new segments of PM. However, electron microscopy studies have clearly shown that the PM incorporated at the tip greatly exceeds elongation and a mechanism of PM retrieval was already postulated in the mid-nineteenth century. Recent studies on endocytosis during pollen tube growth showed that different endocytic pathways occurred in distinct zones of the tube, including the apex, and led to a new hypothesis to explain vesicle accumulation at the tip; namely, that endocytic vesicles contribute substantially to V-shaped vesicle accumulation in addition to SVs and that exocytosis does not involve the entire apical domain. New insights suggested the intriguing hypothesis that modulation between exo- and endocytosis in the apex contributes to maintain PM polarity in terms of lipid/protein composition and showed distinct degradation pathways that could have different functions in the physiology of the cell. Pollen tube growth in vivo is closely regulated by interaction with style molecules. The study of endocytosis and membrane recycling in pollen tubes opens new perspectives to studying pollen tube-style interactions in vivo.     

A Calcium-Dependent Protein Kinase Interactswith and Activates A Calcium Channel toRequlate Pollen Tube Growth

ABSTRACT Calcium, as a ubiquitous second messenger, plays essential roles in tip-growing cells, such as animal neu-rons, plant pollen tubes, and root hairs. However, little is known concerning the regulatory mechanisms that code anddecode Ca2＋ signals in plants. The evidence presented here indicates that a calcium-dependent protein kinase, CPK32,controls polar growth of pollen tubes. Overexpression of CPK32 disrupted the polar growth along with excessive Ca2＋accumulation in the tip. A search of downstream effector molecules for CPK32 led to identification of a cyclic nucleotide-gated channel, CNGC18, as an interacting partner for CPK32. Co-expression of CPK32 and CNGC18 resulted in activationof CNGC18 in Xenopus oocytes where expression of CNGC18 alone did not exhibit significant calcium channel activity.Overexpression of CNGC18 produced a growth arrest phenotype coupled with accumulation of calcium in the tip, simi-lar to that induced by CPK32 overexpression. Co-expression of CPK32 and CNGC18 had a synergistic effect leading tomore severe depolarization of pollen tube growth. These results provide a potential feed-forward mechanism in whichcalcium-activated CPK32 activates CNGC18, further promoting calcium entry during the elevation phase of Ca2＋ oscilla-tions in the polar growth of pollen tubes.     

Airborne Pollen Grains in Zonguldak，Turkey，2001-2002

The variation in airborne pollen concentration of the Zonguldak region．Turkey was studied for two consecutive years 2001-2002 using a Durham sampler．During this period．a total of 61 304 pollen grains belonging to 43 taxa were recorded．Of these 43 taxa．26 belonged to arboreal and 17 to nonarboreal plants．The main pollen types were Pinaceae,Populus,Carpinus,Betula,Corylus,Fagus orientalis,Castanea sativa,AInus glutinosa,Quercus,Cupressaceae,Chenopodiaceae and Gramineae．representing 96．7％of the pollen spectrum．Pollen concentration reached the highest level in March．     

Complete nucleotide sequence and genome organization of tobacco mosaic virus isolated from Viciafaba

Based on reported TMV-U1 sequence, primers were designed and fragments covering the entire genome of TMV broad bean strain (TMV-B) were obtained with RT-PCR. These fragments were cloned and sequenced and the 5' and 3' end sequences of genome were confirmed with RACE. The complete sequence of TMV-B comprises 6 395 nucleotides (nt) and four open reading frames, which correspond to 126 ku (1 116 amino acids), 183 ku (1 616 amino acids), 30 ku (268 amino acids) and 17.5 ku proteins (159 amino acids). The complete nucleotide sequence of TMV-B is 99.4% identical to that of TMV-U1. The two virus isolates share the same sequence of 5', 3' non-coding region and 17.5 K ORF, and 6, 1 and 3 amino acid changes are found in 126 K protein, 54 K protein and 30 K protein, respectively. The possible mechanism on the infection of TMV-B in Vicia faba is discussed.     

Pollen Morphology of Parnassia L. （Parnassiaceae） and Its Systematic Implications

The pollen morphology of 28 species of Parnassia L. was investigated with light microscopy and scanning electron microscopy (SEM). The shape of pollen grains in this genus varies from subspheroidal to prolate in equatorial view and is three-lobed circular in the polar view. Pollen grains are usually radially symmetrical, isopolar, tricolporate or syntricolporate, with reticulate sculpture. The pollen characteristics among species are fairly similar to each other. Morphological information regarding the pollen grains shows that Parnassia is a natural genus. Based on exine ornamentation observed under SEM, three types of pollen grains were recognized: (i) type I, with foveolate-reticulate sculpture; (ii) type II, with a finely reticulate sculpture; and (iii) type III, with a coarsely reticulate sculpture. Most sections of this genus have one type of sculpture of pollen morphology, but Sect. Nectarotrilobos has three types of sculpture and Sect.Saxifragastrum has two types of sculpture. All three types of sculpture can be found in Southwest China,with species with the longest (Parnassia delavayi Franch.) and shortest (Parnassiafaberi Oliv.) colpi,implying that Southwest China is the center of diversification of the genus.     

RNA-binding domain of the key structural protein P7 for the Rice dwarf virus particle assembly

The Rice dwarf virus (RDV) P7 structural protein is the key protein in the RDV particle assembly. The P7 protein was digested partially or completely by Staphylococcus aureus V8 protease and/or Pseudomonasfragi Asp-N protease. The molecular mass and the N-terminal amino acid sequence of the polypeptide fragments of the P7 protein were determined by SDS-PAGE and the Edman degradation method, respectively. Then the polypeptides were located in the deduced amino acid sequence of the RDV P7 protein based on the nucleotide sequence information, with the knowledge of the specific cleavage sites of the Staphylococcus aureus V8 and Pseudomonasfragi Asp-N protease, and the two RNA-binding domains in the P7 protein were identified. Domain 1 was located in the residue 128-249 containing 122 amino acids and domain 2 was located in the residue 325-355 containing 31 amino acids. Thus, these two domains may play an important role in the virus particle assembly by contributing to the packaging of viral dsRNAs inside the particles. The two domains may be novel RNA-binding domains, because no amino acid sequences highly similar to the conservative sequences of known dsRNA-binding domains reported so far. The similarity between the motif of domain 1 and the motif of the DNA-binding protein suggests that the DNA-binding activity of the RDV P7 protein may be due to this sequence. The similarity between the motif of domain 1 and the motif of the RNA polymerase domain suggests that the P7 protein may also play a role in RNA synthesis, besides its function in the assembly and subsequent packaging of viral dsRNA into core particles.     

Predicting protein subcellular location using digital signal processing

The biological functions of a protein are closely related to its attributes in a cell. With the rapid accumulation of newly found protein sequence data in databanks, it is highly desirable to develop an automated method for predicting the subcellular location of proteins. The establishment of such a predictor will expedite the functional determination of newly found proteins and the process of prioritizing genes and proteins identified by genomic efforts as potential molecular targets for drug design. The traditional algorithms for predicting these attributes were based solely on amino acid composition in which no sequence order effect was taken into account. To improve the prediction quality, it is necessary to incorporate such an effect. However, the number of possible patterns in protein sequences is extremely large, posing a formidable difficulty for realizing this goal. To deal with such difficulty, a well-developed tool in digital signal processing named digital Fourier transform (DFT) [1] was introduced. After being translated to a digital signal according to the hydrophobicity of each amino acid, a protein was analyzed by DFT within the frequency domain. A set of frequency spectrum parameters, thus obtained, were regarded as the factors to represent the sequence order effect. A significant improvement in prediction quality was observed by incorporating the frequency spectrum parameters with the conventional amino acid composition. One of the crucial merits of this approach is that many existing tools in mathematics and engineering can be easily applied in the predicting process. It is anticipated that digital signal processing may serve as a useful vehicle for many other protein science areas.     

GNOM-LIKE 2, Encoding an Adenosine Diphosphate-Ribosylation Factor-Guanine Nucleotide Exchange Factor Protein Homologous to GNOM and GNL1, is Essential for Pollen Germination in Arabidopsis

In flowering plants, male gametes are delivered to female gametophytes by pollen tubes. Although it is important for sexual plant reproduction, little is known about the genetic mechanism that controls pollen germination and pollen tube growth. Here we report the identification and characterization of two novel mutants, gnom-like 2-1 （gnl2-1） and gn12-2 in Arabidopsis thaliana, in which the pollen grains failed to germinate in vitro and in vivo. GNL2 encodes a protein homologous to the adenosine diphosphate-ribosylation factor-guanine nucleotide exchange factors, GNOM and GNL1 that are involved in endosomal recycling and endoplasmic reticulum-Golgi vesicular trafficking. It was prolifically expressed in pollen grains and pollen tubes. The results of the present study suggest that GNL2 plays an important role in pollen germination.     

Airborne Pollen Grains of Bozuyuk （Bilecik, Turkey）

Airborne pollen grains in the atmosphere of Bozuyuk were investigated over a 2 yr period from 2000 to 2001 using a Durham sampler. A total number of pollen grains of 5 170 pollen grains belonging to 32 taxa were identified and recorded along with some unidentified pollen grains. Of all the pollen grains, 78.66% were arboreal, 19.20% were non-arboreal, and 2.12% were unidentified. The majority of pollen grains investigated were Pinus, Platanus, Quercus, Cupressaceae, Poaceae, Fagus, Salix, Rosaceae, Urticaceae, Asteraceae, and Chenopodiaceae. The maximum number of pollen grains was recorded in May.     

The Functional Motif of SARS-CoV S Protein Involved in the Interaction with ACE2

SARS-CoV is a newly discovery pathogen causing severe acute respiratory problems. It has been established that the S protein in this pathogen plays an important rule in the adsorption and penetration of SARS-CoV into the host cell by interaction with the ACE2 receptor. To determinant which functional motif of the S protein was involved in the interaction with ACE2, seven truncated S proteins deleted from the N or C terminal were obtained by an E.coli expression system and purified by column chroma-tography to homogeneity. Each truncated S protein was fixed on to the well of an ELISA plate and an interaction was initiated with the ACE2 protein. The adsorption were quantified by ELISA, and the results indicated that amino acids from 388 to 496 of the S protein was responsible for the interaction with the ACE2 receptor, and the interaction could be completely disrupted by an antibody specific to these amino acids. Deletions adjacent to this domain did not appear to have a significant impact on the interaction with ACE2, suggesting that the S protein of SARS-CoV could be developed as a vaccine to prevent the spread of SARS-CoV.     

A Putative Calcium-Permeable Cyclic Nucleotide-Gated Channel, CNGC18, Regulates Polarized Pollen Tube Growth

A tip-focused Ca^2＋ gradient is tightly coupled to polarized pollen tube growth, and tip-localized influxes of extracellular Ca^2＋ are required for this process. However the molecular identity and regulation of the potential Ca^2＋ channels remains elusive. The present study has implicated CNGC18 （cyclic nucleotide-gated channel 18） in polarized pollen tube growth, because its overexpression induced wider and shorter pollen tubes. Moreover, CNGC18 overexpression induced depolarization of pollen tube growth was suppressed by lower extracellular calcium （[Ca^2＋]ex）. CNGC18-yellow fluorescence protein （YFP） was preferentially localized to the apparent post-Golgi vesicles and the plasma membrane （PM） in the apex of pollen tubes. The PM localization was affected by tip-localized ROP1 signaling. Expression of wild type ROP1 or an active form of ROP1 enhanced CNGC18-YFP localization to the apical region of the PM, whereas expression of RopGAP1 （a ROP1 deactivator） blocked the PM localization. These results support a role for PM-Iocalized CNGC18 in the regulation of polarized pollen tube growth through its potential function in the modulation of calcium influxes.