Tobacco Pollen Research Articles

Tobacco pollen: archaeological and forensic applications

The fossil pollen contents found in the charred dottle residue from 20 clay pipe bowls recovered from a cesspit at a house in Amsterdam, the Netherlands (which was owned and used by the artist Rembrandt Harmenszoon van Rijn), were collected and studied. The clay pipes date from approximately the same period when Rembrandt lived in the house and used the cesspit. We believe that some of the pollen in the dottle residue may have come from the tobacco, some possibly from airborne sources at Rembrandt's house and/or from debris in the cesspit in which the pipes were discarded. The results are compared with a similar study of pipe dottle from Native American clay pipes conducted more than 25 years ago. We also briefly review the origin and spread of domestic tobacco in an effort to determine potential sources of the tobacco imported and used in the Netherlands during the lifetime of Rembrandt and the period when he occupied the house. The pollen content of modern brands of commercial pipe tobacco is studied and the potential for using pollen found in tobacco products as evidence in forensic circumstances is reviewed.

Defensin (TvD1) from Tephrosia villosa exhibited strong anti-insect and anti-fungal activities in transgenic tobacco plants

Plant defensin is a small, cationic, cysteine-rich broad-spectrum antimicrobial peptide with four or five disulfide bridges and has been shown to be a component of the innate immunity system in plants. In the present study, the defensin gene (TvD1) from Tephrosia villosa was overexpressed in tobacco and characterized. Two high-expression (T1, T26) and one low-expression (T13) plant lines were selected through semi-quantitative RT-PCR and used for bioassays along with non-transgenic controls. The high-expression plant line exhibited strong in vivo anti-fungal and anti-feedant activity against the pathogen Rhizoctonia solani and the first and second instar larvae of the Spodoptera litura (F.), the tobacco cutworm, respectively. The low-expression plant line showed a moderate level of tolerance/resistance in the bioassays. The recombinant peptide (rTvD1) exhibited toxicity to tobacco pollen grains in the germination assay, but transgenic plants produced copious fertile pollen and set capsules with viable seeds. The results of this study demonstrate that the single gene (TvD1) effectively controls both fungal and insect pests and, hence, it can be used for crop transformation.

Revealing phosphoproteins playing role in tobacco pollen activated in vitro

The transition between the quiescent mature and the metabolically active germinating pollen grain most probably involves changes in protein phosphorylation status, since phosphorylation has been implicated in the regulation of many cellular processes. Given that, only a minor proportion of cellular proteins are phosphorylated at any one time, and that phosphorylated and nonphosphorylated forms of many proteins can co-exist within a cell, the identification of phosphoproteins requires some prior enrichment from a crude protein extract. Here, we have used metal oxide/hydroxide affinity chromatography (MOAC) based on an aluminum hydroxide matrix for this purpose, and have generated a population of phosphoprotein candidates from both mature and in vitro activated tobacco pollen grains. Both electrophoretic and nonelectrophoretic methods, allied to MS, were applied to these extracts to identify a set of 139 phosphoprotein candidates. In vitro phosphorylation was also used to validate the spectrum of phosphoprotein candidates obtained by the MOAC phosphoprotein enrichment. Since only one phosphorylation site was detected by the above approach, titanium dioxide phosphopeptide enrichment of trypsinized mature pollen crude extract was performed as well. It resulted in a detection of additional 51 phosphorylation sites giving a total of 52 identified phosphosites in this set of 139 phosphoprotein candidates.

Relation between changes in polyamine, protective enzyme activity and pollen vigor of tobacco in different flowering stages

Pollen vigor is a prerequisite for fertilization and seed development. In the present research, the pollen of two tobacco varieties of Yunyan87 and K236 were collected in four different flowering stages. Pollen germination rate and the length of pollen tube were tested. Nine physiological and biochemical parameters of putrescine (Put), spermidine (Spd), spermine (Spm), superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), malondialdehyde (MDA) and relative electrolyte leakage (REL) were examined. Multiple linear regression was adopted to determine the relationship between all parameters and pollen vigor. The results suggested that Spd could be used as a valuable parameter to predict pollen vigor for Yunyan87 and MDA could be used as a valuable parameter to predict pollen vigor for K236 in different flowering stages. The results also suggested that tobacco pollen collected in petals unfurling stage and full blooming stage had the highest vigor and unfurling stage was the best stage for pollen collection. Key words: Pollen vigor, polyamine, antioxidant enzyme, pollen germination.

Ni2+ effects on Nicotiana tabacum L. pollen germination and pollen tube growth

To investigate the mechanisms of Ni(2+) effects on initiation and maintenance of polar cell growth, we used a well-studied model system-germination of angiosperm pollen grains. In liquid medium tobacco pollen grain forms a long tube, where the growth is restricted to the very tip. Ni(2+) did not prevent the formation of pollen tube initials, but inhibited their subsequent growth with IC(50) = 550 μM. 1 mM Ni(2+) completely blocked the polar growth, but all pollen grains remained viable, their respiration was slightly affected and ROS production did not increase. Addition of Ni(2+) after the onset of germination had a bidirectional effect on the tubes development: there was a considerable amount of extra-long tubes, which appeared to be rapidly growing, but the growth of many tubes was impaired. Studying the localization of possible targets of Ni(2+) influence, we found that they may occur both in the wall and in the cytoplasm, as confirmed by specific staining. Ni(2+) disturbed the segregation of transport vesicles in the tips of these tubes and significantly reduced the relative content of calcium in the aperture area of pollen grains, as measured by X-ray microanalysis. These factors are considered being critical for normal polar cell growth. Ni(2+) also causes the deposition of callose in the tips of the tube initials and the pollen tubes that had stopped their growth. We can assume that Ni(2+)-induced disruption of calcium homeostasis can lead to vesicle traffic impairment and abnormal callose deposition and, consequently, block the polar growth.

Transmembrane transport of K+ and Cl− during pollen grain activation in vivo and in vitro

We studied the possibility of K+ and Cl- efflux from tobacco pollen grains during their activation in vitro or on the stigma of a pistil. For this purpose the X-ray microanalysis and spectrofluorometry were applied. We found that the relative content of potassium and chlorine in the microvolume of pollen grain decreases during its hydration and activation on stigma. Efflux of these ions was found both in vivo and in vitro. In model in vitro experiments anion channel inhibitor NPPB ((5-nitro-2-(3-phenylpropylamino) benzoic acid) in the concentration that was blocking pollen germination, reduced Cl- efflux; potassium channel inhibitor (tetraethylammonium chloride) partially reduced K+ efflux and lowered the percent of activated cells. Another blocker of potassium channels Ba2+ caused severe decrease in cell volume and blocked the activation. In general, the obtained data demonstrates that the initiation of pollen germination both in vivo and in vitro involves the activation of K+ and Cl- release. An important role in these processes is played by NPPB-, TEA- and Ba(2+)-sensitive plasmalemma ion channels.

Inhibition of actin polymerisation by low concentration Latrunculin B affects endocytosis and alters exocytosis in shank and tip of tobacco pollen tubes

Pollen tube growth depends on the integrity of the actin cytoskeleton that regulates cytoplasmic streaming and secretion. To clarify whether actin also plays a role in pollen tube endocytosis, Latrunculin B (LatB) was employed in internalisation experiments with tobacco pollen tubes, using the lipophilic dye FM4-64 and charged nanogold. Time-lapse analysis and dissection of endocytosis allowed us to identify internalisation pathways with different sensitivity to LatB. Co-localisation experiments and ultrastructural observations using positively charged nanogold revealed that LatB significantly inhibited endocytosis in the pollen tube shank, affecting internalisation of the plasma membrane (PM) recycled for secretion, as well as that conveyed to vacuoles. In contrast, endocytosis of negatively charged nanogold in the tip, which is also conveyed to vacuoles, was not influenced. Experiments of fluorescence recovery after photobleaching (FRAP) of the apical and subapical PM revealed domains with different rates of fluorescence recovery and showed that these differences depend on the actin cytoskeleton integrity. These results show the presence of distinct degradation pathways by demonstrating that actin-dependent and actin-indepedent endocytosis both operate in pollen tubes, internalising tracts of PM to be recycled and broken down. Intriguingly, although most studies concentrate on exocytosis and distension in the apex, the present paper shows that uncharacterised, actin-dependent secretory activity occurs in the shank of pollen tubes.

Maize transcription factor Zmdof1 involves in the regulation of Zm401 gene

Zmdof1 is a member of the maize Dof transcription factor family genes and participates in the regulation and control of the PEPC gene. The Zm401 gene, which contains short open reading frames (ORFs), has been cloned from maize, and its promoter contains several Zmdof1 recognition sites (DOFCORE, AAAG). Zm401 has an important role in anther development, and the protein encoded by the longest ORF, Zm401p10, localizes in the nucleus and is essential for maize anther development. In this study, we cloned Zmdof1, and expression pattern assay suggested that Zmdof1 has a role not only in nutrition organ development but also in maize pollen maturation. Transient expression of a Zmdof1::GFP fusion protein in onion epidermal cells showed a nuclear localization. 5′ deletion analysis of the Zm401 promoter showed that the region of −670 to −510 is important for promoter activity. Trans-activation assays in the yeast one-hybrid system confirmed that Zmdof1 had a strong interaction with AAAG elements in the Zm401 promoter. Co-transformation of a PZm401::Gus construct with UBI::Zmdof1 resulted in an approximately 40% decrease in GUS expression. Decrease of pollen viability resulting from ectopic expression of Zm401 controlled by its native promoter was recovered when Zmdof1 was transformed. Quantitative RT-PCR analysis showed that in PZm401::Zm401/UBI::Zmdof1 transgenic tobacco, the expression of Zm401 decreased significantly, coupled with an increase of Zmdof1 expression. The results indicated that Zmdof1 interacts with the Zm401 promoter in vitro and downregulates Zm401 in transgenic tobacco pollen. A probable regulatory mechanism of Zmdof1 to Zm401 in pollen was proposed.

Turnover of Phosphatidic Acid through Distinct Signaling Pathways Affects Multiple Aspects of Pollen Tube Growth in Tobacco

Phosphatidic acid (PA) is an important intermediate in membrane lipid metabolism that acts as a key component of signaling networks, regulating the spatio-temporal dynamics of the endomembrane system and the cytoskeleton. Using tobacco pollen tubes as a model, we addressed the signaling effects of PA by probing the functions of three most relevant enzymes that regulate the production and degradation of PA, namely, phospholipases D (PLD), diacylglycerol kinases (DGKs), and lipid phosphate phosphatases (LPPs). Phylogenetic analysis indicated a highly dynamic evolution of all three lipid-modifying enzymes in land plants, with many clade-specific duplications or losses and massive diversification of the C2-PLD family. In silico transcriptomic survey revealed increased levels of expression of all three PA-regulatory genes in pollen development (particularly the DGKs). Using specific inhibitors we were able to distinguish the contributions of PLDs, DGKs, and LPPs into PA-regulated processes. Thus, suppressing PA production by inhibiting either PLD or DGK activity compromised membrane trafficking except early endocytosis, disrupted tip-localized deposition of cell wall material, especially pectins, and inhibited pollen tube growth. Conversely, suppressing PA degradation by inhibiting LPP activity using any of three different inhibitors significantly stimulated pollen tube growth, and similar effect was achieved by suppressing the expression of tobacco pollen LPP4 using antisense knock-down. Interestingly, inhibiting specifically DGK changed vacuolar dynamics and the morphology of pollen tubes, whereas inhibiting specifically PLD disrupted the actin cytoskeleton. Overall, our results demonstrate the critical importance of all three types of enzymes involved in PA production and degradation, with strikingly different roles of PA produced by the PLD and DGK pathways, in pollen tube growth.

Structural characterization, solution stability, and potential health and environmental effects of the Nano-TiO2 bioencapsulation matrix and the model product of its biodegradation TiBALDH

Dispersions of small TiO2 nanoparticles in alcohol, stabilized by surface complexation with protonated triethanolamine ligands, are proposed as bio-encapsulation matrices. They were characterized by TEM and EXAFS spectroscopy, and DLS was used to investigate the nanoparticles stability over time in their pure form and after insertion into aqueous medium. This study reveals unusual structural features that explain the recently demonstrated facile formation of dense encapsulates on the surface of biological objects. In the view of the potentially broad application of this already industrially available material in agriculture as an encapsulation matrix for biocontrol organisms, its potential health and environmental effects were characterized by employing a number of model systems. The potential health effects of the produced stable aqueous dispersions were studied in vitro using A549 and U1810 lung carcinoma cell lines. The nanotitania in the environment is partly bio-digested with the formation of citrate and lactate complexes. The effects on the growth of tobacco pollen grains by the nanotitania and of the ammonium lactato-oxo-titanate (TiBALDH, a product already broadly used in biomineralization studies) were investigated to gain insight into the impact of these materials on the environment and specifically on plant reproduction. TiBALDH was used as a model product of the bio-digestion and its structure has been probed in this work by X-ray powder diffraction and EXAFS spectroscopy. No acute negative bio-effects could be observed for the studied materials at significantly high concentrations, such as 50 μg ml−1 for the viability of human lung cancer cells and up to about 120 μg ml−1 for the growth of pollen grains, corresponding to the conditions of proposed field applications. This observation was contrasted by the apparently high toxicity of the LaAlO3 based nanophosphor which was applied as a positive control.

Variable Regions of PI4P 5-Kinases Direct PtdIns(4,5)P2 Toward Alternative Regulatory Functions in Tobacco Pollen Tubes

The apical plasma membrane of pollen tubes contains different PI4P 5-kinases that all produce phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] but exert distinct cellular effects. In the present example, overexpression of Arabidopsis AtPIP5K5 or tobacco NtPIP5K6-1 caused growth defects previously attributed to increased pectin secretion. In contrast, overexpression of Arabidopsis AtPIP5K2 caused apical tip swelling implicated in altering actin fine structure in the pollen tube apex. AtPIP5K5, NtPIP5K6-1, and AtPIP5K2 share identical domain structures. Domains required for correct membrane association of the enzymes were identified by systematic deletion of N-terminal domains and subsequent expression of fluorescence-tagged enzyme truncations in tobacco pollen tubes. A variable linker region (Lin) contained in all PI4P 5-kinase isoforms of subfamily B, but not conserved in sequence, was recognized to be necessary for correct subcellular localization of AtPIP5K5, NtPIP5K6-1, and AtPIP5K2. Deletion of N-terminal domains including the Lin domain did not impair catalytic activity of recombinant AtPIP5K5, NtPIP5K6-1, or AtPIP5K2 in vitro; however, the presence of the Lin domain was necessary for in vivo effects on pollen tube growth upon overexpression of truncated enzymes. Overexpression of catalytically inactive variants of AtPIP5K5, NtPIP5K6-1, or AtPIP5K2 did not influence pollen tube growth, indicating that PtdIns(4,5)P2 production rather than structural properties of PI4P 5-kinases was relevant for the manifestation of growth phenotypes. When Lin domains were swapped between NtPIP5K6-1 and AtPIP5K2 and the chimeric enzymes overexpressed in pollen tubes, the chimeras reciprocally gained the capabilities to invoke tip swelling or secretion phenotypes, respectively. The data indicate that the Lin domain directed the enzymes into different regulatory contexts, possibly contributing to channeling of PtdIns(4,5)P2 at the interface of secretion and actin cytoskeleton.

Wall architecture with high porosity is established at the tip and maintained in growing pollen tubes of Nicotiana tabacum

A major question in pollen tube growth in planta remains: do the pollen tube walls form a barrier to interaction with the environment? Using cryo-FESEM, we directly assessed the 3D construction and porosity of tobacco pollen tube walls. Fractured mature primary walls showed a 40-50 nm spaced lattice of continuous fibers interconnected by short rods in the primary wall. These observations agree with TEM observations of sectioned walls. In the secondary callose wall, for which no structure is visible using TEM, cryo-FESEM also revealed a 50 nm lattice consisting of longer fibers, approximately 10-15 nm wide, with rod-like, thinner interconnections at angles of approximately 90° with the longer fibers. Such architecture may reflect functional needs with respect to porosity and mechanical strength. The wall does not form a mechanical barrier to interaction with the environment and is gained at low cost. Cryo-FESEM additionally revealed another special feature of the wall: the tubes were tiled with scales or rings that were highly conspicuous after pectin extraction with EDTA. These rings cause the typical banding patterns of pectin that are commonly seen in pollen tubes during oscillatory growth, as confirmed by staining with toluidine blue as well as by DIC microscopy. Growth analysis by VEC-LM showed that the ring- or scale-like structures of the primary wall consist of material deposited prior to the growth pulses. The alternating band pattern seen in the callose wall is probably imposed by constrictions resulting from the rings of the primary wall.

Vacuolar Sorting Receptor (VSR) Proteins Reach the Plasma Membrane in Germinating Pollen Tubes

Vacuolar sorting receptors (VSRs) are type I integral membrane proteins that mediate the vacuolar transport of soluble cargo proteins via prevacuolar compartments (PVCs) in plants. Confocal immunofluorescent and immunogold Electron Microscope (EM) studies have localized VSRs to PVCs or multivesicular bodies (MVBs) and trans-Golgi network (TGN) in various plant cell types, including suspension culture cells, root cells, developing and germinating seeds. Here, we provide evidence that VSRs reach plasma membrane (PM) in growing pollen tubes. Both immunofluorescent and immunogold EM studies with specific VSR antibodies show that, in addition to the previously demonstrated PVC/MVB localization, VSRs also localize to PM in lily and tobacco pollen tubes prepared from chemical fixation or high-pressure freezing/frozen substitution. Such a PM localization suggests an additional role of VSR proteins in mediating protein transport to PM and endocytosis in growing pollen tubes. Using a high-speed Spinning Disc Confocal Microscope, the possible fusion between VSR-positive PVC organelles and the PM was also observed in living tobacco pollen tubes transiently expressing the PVC reporter GFP-VSR. In contrast, the lack of a prominent PM localization of GFP-VSR in living pollen tubes may be due to the highly dynamic situation of vesicular transport in this fast-growing cell type.

Impact of homogenization and protein extraction conditions on the obtained tobacco pollen proteomic patterns

Mature pollen grain represents a highly desiccated structure with an extremely tough cell wall. Thanks to it, it resists common proteomic protocols. Instead, a robust homogenization has to be performed since proteins are needed to burst out of the cell to be included in the extracted proteome fraction. Here, a novel way of pollen homogenization employing Roche MagNA Lyser Instrument is presented, sparing time and laborious work. However, plant proteomics does not rely solely on perfect homogenization; also the choice of the extraction protocol is of key importance. The composition of the extraction buffer has a decisive influence on which proteome fraction will be extracted. Therefore the second part of our study is dedicated to the comparison of different extraction protocols with respect to subsequent proteomic analyses.

Impact of homogenization and protein extraction conditions on the obtained tobacco pollen proteomic patterns

Mature pollen grain represents a highly desiccated structure with an extremely tough cell wall. Thanks to it, it resists common proteomic protocols. Instead, a robust homogenization has to be performed since proteins are needed to burst out of the cell to be included in the extracted proteome fraction. Here, a novel way of pollen homogenization employing Roche MagNA Lyser Instrument is presented, sparing time and laborious work. However, plant proteomics does not rely solely on perfect homogenization; also the choice of the extraction protocol is of key importance. The composition of the extraction buffer has a decisive influence on which proteome fraction will be extracted. Therefore the second part of our study is dedicated to the comparison of different extraction protocols with respect to subsequent proteomic analyses.

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 phosphomimetic mutation of an evolutionarily conserved serine residue affects the signaling properties of Rho of plants (ROPs)

Plant ROP (Rho of plants) proteins form a unique subgroup within the family of Rho-type small G-proteins of eukaryotes. In this paper we demonstrate that the phosphomimetic mutation of a serine residue conserved in all Rho proteins affects the signaling properties of plant ROPs. We found that the S74E mutation in Medicago ROP6 and Arabidopsis ROP4 prevented the binding of these proteins to their plant-specific upstream activator the plant-specific ROP nucleotide exchanger (PRONE)-domain-containing RopGEF (guanine nucleotide exchange factor) protein and abolished the PRONE-mediated nucleotide exchange reaction in vitro. Structural modeling supported the hypothesis that potential phosphorylation of the S74 residue interferes with the binding of the PRONE-domain to the adjacent plant-specific R76 residue which plays an important role in functional ROP-PRONE interaction. Moreover, we show that while the binding of constitutively active MsROP6 to the effector protein RIC (ROP-interactive CRIB-motif-containing protein) was not affected by the S74E mutation, the capability of this mutated protein to bind and activate the RRK1 kinase in vitro was reduced. These observations are in agreement with the morphology of tobacco pollen tubes expressing mutant forms of yellow fluorescent protein (YFP):MsROP6. The S74E mutation in MsROP6 had no influence on pollen tube morphology and attenuated the phenotype of a constitutively active form of MsROP6. The presented Medicago and Arabidopsis data support the notion that the phosphorylation of the serine residue in ROPs corresponding to S74 in Medicago ROP6 could be a general principle for regulating ROP activation and signaling in plants.

A Tobacco Homolog of DCN1 is Involved in Cellular Reprogramming and in Developmental Transitions

AbstractPlant proteomes show remarkable plasticity in reaction to environmental challenges and during developmental transitions. Some of this adaptability comes from ubiquitin-mediated protein destruction regulated by cullin-RING E3 ubiquitin ligases (CRLs). CRLs are activated through modification of the cullin subunit with the ubiquitin-like protein RUB/NEDD8 by an E3 ligase called defective in cullin neddylation 1 (DCN1). Here we show that tobacco DCN1 binds ubiquitin and RUB/NEDD8, and associates with cullin. When knocked down by RNAi, tobacco pollen formation stopped and zygotic embryogenesis was blocked around the globular stage. Additionally, we found that RNAi of DCN1 inhibited the stress-triggered reprogramming of cultured microspores from their intrinsic gametophytic mode of development to an embryogenic state. This stress-induced developmental switch is a known feature in many important crops and leads ultimately to the formation of haploid embryos and plants. Compensating the RNAi effect by re-transformation with a promoter-silencing construct restored pollen development and zygotic embryogenesis, as well as the ability for stress-induced formation of embryogenic microspores. Overexpression of DCN1, however, accelerated pollen tube growth and increased the potential for microspore reprogramming. These results demonstrate that the biochemical function of DCN1 is conserved in plants and that its activity is specifically required for transitions during pollen development and embryogenesis, and for pollen tube tip growth.

Mutations in Two Putative Phosphorylation Motifs in the Tomato Pollen Receptor Kinase LePRK2 Show Antagonistic Effects on Pollen Tube Length

The tip-growing pollen tube is a useful model for studying polarized cell growth in plants. We previously characterized LePRK2, a pollen-specific receptor-like kinase from tomato (1). Here, we showed that LePRK2 is present as multiple phosphorylated isoforms in mature pollen membranes. Using comparative sequence analysis and phosphorylation site prediction programs, we identified two putative phosphorylation motifs in the cytoplasmic juxtamembrane (JM) domain. Site-directed mutagenesis in these motifs, followed by transient overexpression in tobacco pollen, showed that both motifs have opposite effects in regulating pollen tube length. Relative to LePRK2-eGFP pollen tubes, alanine substitutions in residues of motif I, Ser(277)/Ser(279)/Ser(282), resulted in longer pollen tubes, but alanine substitutions in motif II, Ser(304)/Ser(307)/Thr(308), resulted in shorter tubes. In contrast, phosphomimicking aspartic substitutions at these residues gave reciprocal results, that is, shorter tubes with mutations in motif I and longer tubes with mutations in motif II. We conclude that the length of pollen tubes can be negatively and positively regulated by phosphorylation of residues in motif I and II respectively. We also showed that LePRK2-eGFP significantly decreased pollen tube length and increased pollen tube tip width, relative to eGFP tubes. The kinase activity of LePRK2 was relevant for this phenotype because tubes that expressed a mutation in a lysine essential for kinase activity showed the same length and width as the eGFP control. Taken together, these results suggest that LePRK2 may have a central role in pollen tube growth through regulation of its own phosphorylation status.

An ultrastructural study of cytomixis in tobacco pollen mother cells

Intercellular chromatin migration (cytomixis) in the pollen mother cells of two tobacco (Nicotiana tabacum L.) lines was analyzed by electron microscopy during the first meiotic prophase. The maximal manifestation of cytomixis was observed in the pachytene. As a rule, several cells connected with one another by cytomictic channels wherein the nuclei migrated were observable at this stage. In the majority of cases, nuclei passed from cell to cell concurrently through several closely located cytomictic channels. Chromatin migrated between cells within the nuclear envelope, and its disintegration was unobservable. The nucleus, after passing through cytomictic channels into another cell, can be divided into individual micronuclei or, in the case of a direct contact with another nucleus, can form a nuclear bridge. It has been demonstrated that the chromatin structure after intracellular migration visually matches the chromatin structure before it passed through the cytomictic channel. No signs of pyknosis were observable in the chromatin of the micronuclei formed after cytomixis, and the synaptonemal complex was distinctly seen. The dynamics of changes in the nucleoli during cytomixis was for the first time monitored on an ultrastructural level. Possible mechanisms determining cytomixis are discussed and the significance of this process in plant development is considered.