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A functional study of stylar hydroxyproline-rich glycoprotein during pollen tube growth
Abstract
Class III pistil-specific extensin-like proteins (PELPIII) are chimeric hydroxyproline-rich glycoproteins with properties of both extensins and arabinogalactan proteins. The abundance and specific localization of PELPIII in the intercellular matrix (IM) of tobacco (Nicotiana tabacum) stylar transmitting tissue, and translocation of PELPIII from the IM into the pollen tube wall after pollination, presume the biological function of these glycoproteins to be related to plant reproduction. Here we show that in in vitro assays the translocation of PELPIII is specifically directed to the callose inner wall of the pollen tubes, indicating that protein transfer is not dependent on the physiological conditions of the transmitting tract. We designed a set of experiments to elucidate the biological function of PELPIII in the stylar IM. To study the function of the specific interaction between PELPIII proteins and the pollen tube wall, one of the PELPIII proteins (MG15) was ectopically expressed in pollen tubes and targeted to the tube wall. We also generated transgenic tobacco plants in which PELPIII proteins were silenced. In vitro bioassays were performed to test the influence of purified PELPIII on pollen tube growth, as compared to tobacco transmitting tissue-specific proteins (TTS) that were previously shown to stimulate pollen tube growth. The various tests described for activity of PELPIII proteins all gave consistent and mutually affirmative results: the biological function of PELPIII proteins is not directly related to pollen tube growth. These data show that similar stylar glycoproteins may act very differently on pollen tubes.
... To determine whether PELPIII functions in regulating PTG [11] produced a PELPIII-antisense N. tabacum line with undetectable levels of PELPIII AGP and pollinated it with N. tabacum, N. rustica or N. maritima pollen. No differences in PTG between the antisense lines and normal plants occurred. ...
... Reduction of endogenous PEL-PIII (S plus T) mRNA levels in the RNAiPIII transgenic line resulted in increased N. obtusifolia PTG relative to normal styles but did not alter PTG of thirteen other Nicotiana species, including N. repanda (Additional file 1). It was previously shown that pollination of styles of antisense plants with reduced PELPIII mRNA levels in N. tabacum 'Petite Havana' SR1 resulted in longer PTG of both N. obtusifolia and N. repanda relative to normal 'Petite Havana' SR1styles [10,11]. Our results were similar to those of Eberle [10] with reduced PELPIII in the RNAiPIII transgenic line and increased N. obtusifolia PTG (Fig. 4). ...
Background
The transmitting tissue of the style is the pathway for pollen tube growth to the ovules and has components that function in recognizing and discriminating appropriate pollen genotypes. In Nicotiana tabacum, the class III pistil extensin-like (PELPIII) arabinogalactan protein is essential for the inhibition of N. obtusifolia pollen tube growth. The transmitting tissue-specific (TTS) arabinogalactan protein amino acid sequence and expression pattern is similar to PELPIII, but it facilitates self-pollinated N. tabacum. The TTS and PELPIII arabinogalactan protein can be divided into the less conserved N-terminal (NTD) and the more conserved C-terminal (CTD) domains. This research tested whether the NTD is the key domain in determining PELPIII function in the inhibition of interspecific pollen tube growth. Three variant PELPIII gene constructs were produced where the PELPIII NTD was exchanged with the TTS NTD and a single amino acid change (cysteine to alanine) was introduced into the PELPIII NTD. The PELPIII variants of N. tabacum were tested for activity by measuring the inhibition N. obtusifolia pollen tube growth by using them to complement a 3’UTR RNAi transgenic line with reduced PELPIII mRNA.
Results
The RNAi N. tabacum line had reduced PELPIII mRNA accumulation and reduced inhibition of N. obtusifolia pollen tube growth, but had no effect on self-pollen tube growth or pollen tube growth of 12 other Nicotiana species. The NTD of PELPIII with either the PELPIII or TTS CTDs complemented the loss PELPIII activity in the RNAi transgenic line as measured by inhibition of N. obtusifolia pollen tube growth. The TTS NTD with the PELPIII CTD and a variant PELPIII with a cysteine to alanine mutation in its NTD failed to complement the loss of PELPIII activity and did not inhibit N. obtusifolia pollen tube growth.
Conclusion
The NTD is a key determinant in PELPIII’s function in regulating interspecific pollen tube growth and is a first step toward understanding the mechanism of how PELPIII NTD regulates pollen tube growth.
... Overexpression of MtZpt2-1 in roots conferred salt tolerance and affected the expression of three putative targets in the predicted manner: a cold-regulated A (CORA) homolog, a flower-promoting factor (FPF1) homolog and an auxin-induced proline-rich protein (PRP) gene (Merchan et al. 2007). High contents of hydroxyproline-rich glycoproteins as well as proline-rich proteins are associated with pollen fertility (Rubinstein et al. 1995, Showalter 2001, Bosch et al. 2003. ...
The seeds of two medicinal plants, namely Chamomilla recutita (Babong) and Origanum majorana (Bardaqoush) were subjected to germination in different NaCl concentrations, polyamines (putrescine, spermidine, spermine) and to combination of both. The results revealed that the growth alterations induced by NaCl were alleviated by various levels of polyamines. The organic solutes of both plant seedlings exhibited somewhat variable responses to various salinity levels or polyamines treatments and in combination of both treatments. Putrescine in Ch. recutita seedlings was more effective in alleviating the stress effects of salinization than spermidine and spermine, while in O. majorana seedlings spermidine was more effective. Generally, the degree of stimulation differed according to the type, concentration of the additive used and the type of the plant tested.
... Compatible interactions are guaranteed by multiple-step pathways based on various factors e.g. cysteine-rich proteins (Mollet et al. 2000), glycoproteins (Bosch and Derksen 2003), pectins (Mollet et al. 2000), flavonoids (Mo et al. 1992), calcium gradients (Zhao and Yang 2003) and gamma amino-butyric acid (Palanivelu 2003). Moreover, our understanding of pollen tube growth, guidance and fertilization has been significantly advanced by the development of techniques for in vitro pollination and fertilization (Janson 1993, Zenkteler 1993, Higashiyama 2001, Vervaeke 2002, Willemse and van Lammeren 2002). ...
Male gametophyte development in higher plants is a complex process that requires the coordinated participation of various cell and tissue types and their associated specific gene expression patterns. The male gametophytic life cycle can be divided into a developmental phase leading to the formation of mature pollen grains, and a functional or progamic phase, beginning with the impact of the grains on the stigma surface and ending at double fertilisation. Pollen ontogeny is also an excellent model in which to dissect the cellular networks that control cell growth, polarity, cellular differentiation and cell signaling. Here we present an overview of important cellular processes in male gametophyte development and recent advances in genetics and genomic approaches that are advancing the field significantly. Genetic approaches have identified a growing number of gametophytic mutants affecting discrete steps during the developmental or progamic phases that are now beginning to uncover some of the key molecular processes involved. With recent technological advances pollen transcriptomic studies now provide the first genome-wide view of male reproductive cell development in Arabidopsis These studies reveal at least two successive global gene expression programs and the identity of a large number of male gametophyte-specific genes and putative transcriptional regulators. Transcriptome analysis has revealed a striking overrepresentation of cell wall metabolism, cytoskeleton and signaling genes in preparation for the progamic phase. This quantum leap in gene-centered knowledge highlights the functional specialization of this pathway and offers many new opportunities for the dissection of cellular processes that control male reproductive success.
... In pollinated styles, PELPIII epitopes are no longer detected in the transmitting tract but found associated with the pollen-tube callose wall and callose plugs (de Graff et al., 2003). Unlike NtTTS, PELPIII is apparently unmodified by the expanding pollen tubes (de Graff et al., 2003) and is not involved in promoting pollen-tube growth and attraction (Bosch et al., 2003). Interestingly, the stylar NaTTS, 120-kDa and PELPIII are able to bind in vitro to S-RNases (Cruz-Garcia et al., 2005), the stylar ribonucleases responsible for the degradation of the pollen-tube rRNA and growth arrest of the S-incompatible pollen in the Solanaceae gametophytic system (McCubbin and Kao, 2000). ...
Background Arabinogalactan proteins (AGPs) are complex proteoglycans of the cell wall found in the entire plant kingdom and in almost
all plant organs. AGPs encompass a large group of heavily glycosylated cell-wall proteins which share common features, including
the presence of glycan chains especially enriched in arabinose and galactose and a protein backbone particularly rich in hydroxyproline
residues. However, AGPs also exhibit strong heterogeneities among their members in various plant species. AGP ubiquity in
plants suggests these proteoglycans are fundamental players for plant survival and development.
... CELP (Cysrich extensin-like protein) has a possible role in the process of pollination and fertilization (Wu et al., 1993). PELPIII (Class III pistil-specific extensin-like proteins) abundantly and specifically localize in the intercellular matrix (IM) of tobacco style transmitting tissues, and translocates from IM to the pollen tube wall after pollination, which indicates that the biological function of the glycoprotein is related to pollen tube growth (Bosch et al., 2003). It was also found that a style-specific 120 kDa protein, which is located in the pistil-grown pollen tubes, was required for S-specific pollen rejection in Nicotiana (Lind et al., 1996;Hancock et al., 2005). ...
Extensins are cell wall basic glycoproteins with a polypeptide backbone that is extremely rich in hydroxyproline. In this
paper, the function of extensins in embryo development was studied in Nicotiana tabacum. By using Western blot and immunohistochemistry, the extensin JIM20 epitopes were found to express in different developmental
stages of embryos, and specifically in the top of the embryo proper (EP) and the suspensor of the late globular embryos. In
order to clarify the functions of extensins, a potent hydroxyproline synthesis inhibitor, 3,4-dehydro-L-proline (3,4-DHP), was used in ovule and embryo culture. The results showed that the addition of 3,4-DHP caused abnormal
embryos with single, asymmetry and supernumerary cotyledon primordia, and continuous culture led to cotyledon defects in the
germinated seedlings. Histological sections showed that the shoot apical meristem (SAM) of the abnormal seedlings was dissimilar
from the controls, especially in the seedlings with cup-shaped cotyledons. Furthermore, the vasculature of the abnormal cotyledons
was in an out-of-order format and contained at least two main veins. Finally, both the hydroxyproline assay and fluorescent
immunolocalization confirmed that 3,4-DHP treatment reduced the level of extensins in the cultured ovules and embryos. These
results indicate that extensins may play important roles in the cotyledon primordium formation, SAM activity, and vasculature
differentiation during embryo development.
... The Class III pistil-specific extensin-like proteins (PELPIII) are chimeric HRGP with properties of both extensins and AGPs. PELPIII is localized in the ECM of tobacco stylar transmitting tissue and was shown to be specifically directed into the callose inner wall of the pollen tubes after pollination (Bosch et al., 2001; Bosch et al., 2003; de Graaf et al., 2004). The Galactose-rich Style Protein (GaRSPG) does not promote pollen tube growth and is localized in the cell walls of the TT (Sommer-Knudsen et al., 1998). ...
The evolutionary success of higher plants relies on a very short gametophytic phase, which underlies the sexual reproduction cycle. Sexual plant reproduction takes place in special organs of the flower: pollen, the male gametophyte, is released from the anthers and then adheres, grows and interacts along various tissues of the female organs, collectively known as the pistil. Finally, it fertilizes the female gametophyte, the embryo sac. Pollen is released as bi or tricellular, highly de-hydrated and presumably containing all the biochemical components and transcripts to germinate. Upon hydration on the female tissues, it develops a cytoplasmic extension, the pollen tube, which is one of the fastest growing cells in nature. Pollen is completely "ready-to-go", but despite this seemingly simple reaction, very complex interactions take place with the female tissues. In higher animals, genetic mechanisms for sex determination establish striking developmental differences between males and females. In contrast, most higher plant species develop both male and female structures within the same flower, allowing self-fertilization. Outcrossing is ensured by self-incompatibility mechanisms, which evolved under precise genetic control, controlling self-recognition and cell-to-cell interaction. Equally important is pollen selection along the female tissues, where interactions between different cell types with inherent signalling properties correspond to check-points to ensure fertilization. Last but not least, pollen-pistil interaction occurs in a way that enables the correct targeting of the pollen tubes to the receptive ovules. In this review, we cover the basic mechanisms underlying sexual plant reproduction, from the structural and cellular determinants, to the most recent genetic advances.
... These glycoproteins are classified as hybrid AGPs because they contain both extensin and AGP motifs (Bosch et al., 2001). Transgenic experiments in which PELP III was suppressed in N. tabacum showed no change in the behavior of the pollen tube even though it integrates into the callose of growing pollen tubes (Bosch et al., 2003;de Graaf et al., 2003). No transgenic experiments investigating the role of 120K in the function of the style have been reported, but immunolocalization studies indicate that 120K enters growing pollen tubes (Lind et al., 1996). ...
S-RNase participates in at least three mechanisms of pollen rejection. It functions in S-specific pollen rejection (self-incompatibility) and in at least two distinct interspecific mechanisms of pollen rejection in Nicotiana. S-specific pollen rejection and rejection of pollen from Nicotiana plumbaginifolia also require additional stylar proteins. Transmitting-tract-specific (TTS) protein, 120 kDa glycoprotein (120K) and pistil extensin-like protein III (PELP III) are stylar glycoproteins that bind S-RNase in vitro and are also known to interact with pollen. Here we tested whether these glycoproteins have a direct role in pollen rejection. 120K shows the most polymorphism in size between Nicotiana species. Larger 120K-like proteins are often correlated with S-specific pollen rejection. Sequencing results suggest that the polymorphism primarily reflects differences in glycosylation, although indels also occur in the predicted polypeptides. Using RNA interference (RNAi), we suppressed expression of 120K to determine if it is required for S-specific pollen rejection. Transgenic SC N. plumbaginifolia x SI Nicotiana alata (S105S105 or SC10SC10) hybrids with no detectable 120K were unable to perform S-specific pollen rejection. Thus, 120K has a direct role in S-specific pollen rejection. However, suppression of 120K had no effect on rejection of N. plumbaginifolia pollen. In contrast, suppression of HT-B, a factor previously implicated in S-specific pollen rejection, disrupts rejection of N. plumbaginifolia pollen. Thus, S-specific pollen rejection and rejection of N. plumbaginifolia pollen are mechanistically distinct, because they require different non-S-RNase factors.
The Nicotiana tabacum transmitting tissue is a highly specialized file of metabolically active cells that is the pathway for pollen tubes from the stigma to the ovules where fertilization occurs. It is thought to be essential to pollen tube growth because of the nutrients and guidance it provides to the pollen tubes. It also regulates gametophytic self-incompatibility in the style. To test the function of the transmitting tissue in pollen tube growth and to determine its role in regulating prezygotic interspecific incompatibility, genetic ablation was used to eliminate the mature transmitting tissue, producing a hollow style. Despite the absence of the mature transmitting tissue and greatly reduced transmitting-tissue-specific gene expression, self-pollen tubes had growth to the end of the style. Pollen tubes grew at a slower rate in the transmitting-tissue-ablated line during the first 24 h post-pollination. However, pollen tubes grew to a similar length 40 h post-pollination with and without a transmitting tissue. Ablation of the N. tabacum transmitting tissue significantly altered interspecific pollen tube growth. These results implicate the N. tabacum transmitting tissue in facilitating or inhibiting interspecific pollen tube growth in a species-dependent manner and in controlling prezygotic reproductive barriers.
Prezygotic interspecific incompatibility (II) involves an active inhibition mechanism between the pollen of one species and the pistil of another. As a barrier to fertilization, II effectively prevents hybridization and maintains species identity. Transgenic ablation of the mature transmitting tract (TT) in Nicotiana tabacum resulted in the loss of inhibition of N. obtusifolia (synonym N. trigonophylla) and N. repanda pollen tube growth. The role of the TT in the II interaction between N. tabacum and N. obtusifolia was characterized by evaluating N. obtusifolia pollen tube growth in normal and TT-ablated N. tabacum styles at different post-pollination times and developmental stages. The II activity of the TT slowed and arrested N. obtusifolia pollen tube growth and was developmentally synchronized. We hypothesized that proteins produced by the mature TT and secreted into the extracellular matrix inhibit II pollen tubes. When extracts from the mature TT of N. tabacum were injected into the TT-ablated style prior to pollination the growth of incompatible pollen tubes of N. obtusifolia and N. repanda was inhibited. The class III pistil-specific extensin-like protein (PELPIII) was consistently associated with specific inhibition of pollen tubes and its requirement for II was confirmed through the use of plants with antisense suppression of PELPIII. Inhibition of N. obtusifolia and N. repanda pollen tube growth required the accumulation of PELPIII in the TT of N. tabacum, supporting PELPIII function in prezygotic II. © 2013 The Authors. The Plant Journal © 2013 Blackwell Publishing Ltd.
The class III pistil-specific extensin-like proteins (PELPIII) of Nicotiana tabacum accumulate in the intercellular matrix (IM) of the style transmitting tissue (TT). After pollination, the 110–140kDa PELPIII is translocated from the IM into the pollen tube walls. PELPIII-like sequences have been found in several solanaceous species. These sequences are expressed in mature non-pollinated styles at both RNA and protein level. Of the genus Nicotiana, the species N. alata, N. x sanderae and N. sylvestris (section Alatae), and N. tomentosiformis and N. otophora (section Tomentosae) showed an expression level of PELPIII homologues similar to that in mature styles of N. tabacum. PELPIII genes were absent in the most ancient species studied, namely N. trigonophylla (section Trigonophyllae). To study the species dependence of the translocation of PELPIII into the pollen tube wall in tobacco, interspecific pollinations on N. tabacum pistils were carried out with pollen from the incongruous species N. rustica, N. trigonophylla and Petunia hybrida, where PELPIII homologues are absent in the style. Immunocytological tests showed that the N. tabacum PELPIII is translocated into the pollen tube walls of all three species. Thus, the pollen tube walls of these species do not form a barrier for IM compounds such as the 110–140kDa PELPIII and the absence of any possible effect of PELPIII on pollen tube growth cannot be due to failure of PELPIII transport through the wall. The importance of these findings is discussed with respect to the evolutionary origin of PELPIII, the pollen pistil interaction, the function of style TT-specific proteins and the physical properties of pollen tube walls.
Sexual plant reproduction requires multiple pollen-pistil interactions from the stigma (pollen adhesion, hydration, and germination) to the ovary (fertilization). Understanding the factors that regulate pollen tube growth is critical to understanding the processes essential to sexual reproduction. Many pollen tube growth assays (PTGAs) have shorter and slower pollen tube growth when compared to pollen tube growth through the style. The identification and study of factors that regulate pollen tube growth have been impeded by a lack of an efficient and reproducible PTGA. The objective of this research is to develop a robust assay for Nicotiana tabacum pollen tube growth in an environment that supports sustained and normal growth yet is amenable to testing the effects of specific factors. In this paper, we introduce a novel PTGA, which uses pistils from N. tabacum that lack a mature transmitting tract (TT) due to tissue-specific ablation. The TT-ablated style supports normal pollen tube growth and the hollow structure of the style allows modification of the growth environment by direct injection of test material. This PTGA is robust and allows for rapid and accurate measurement of pollen tube length and pollen tube morphology, supporting pollen tube growth from 20 to 35°C and at pH ranging from 4.8 to 7.6. Use of the ablated style for a PTGA is a novel method for the culture of pollen tubes with sustained growth in vivo while permitting the application of treatments to the growing pollen tubes.
In this report, the localization and spatial distribution of two categories of pectin, high and low methylesterified, on the background of dynamic in loosely bound calcium (Ca(2+)) in Haemanthus hollow style were studied before and after pollination. In the style transmitting tract of unpollinated pistil, mainly high-methylesterified pectins were present, both in the transmitting tract epidermis and in the style canal. After pollination, an increase in the level of two investigated categories of pectin was observed, but the amount of high-methylesterified one in each period of time analyzed was permanently higher. Locally, in the regions of the style canal penetrated by pollen tubes, process of pectin de-esterification was initiated. However, pollination caused an increase of loosely bound Ca(2+) level in the style transmitting tract, this process appears to be not linked with pectin de-esterification and possible Ca(2+) release after the lysis of Ca(2+) cross-linked de-esterified pectin. Instead, it seems to be based on Ca(2+) exocytosis from the transmitting tract epidermis cells providing a source of Ca(2+) for pollen tubes growing in Haemanthus hollow style.
Changes of glutamate kinase activity (GKA) in plants under cadmium and zinc chronic stress reported here reveal a regulatory role of this enzyme in plant heavy metal stress adaptation and indicate its potential use as a stress biomarker. Results of the first experimental series confirmed the toxic effects of cadmium and zinc at tested levels (30, 60, 90 mg Cd kg(-1) and 250, 500, 750 mg Zn kg(-1) soil) for spinach. A significant decrease of GKA in plants grown on contaminated treatments was found. Changes of GKA of plants grown on the highest contaminated treatments in the second series of experiments showed a similar course as a curve of plant stress response indicating the process of plant adaptation to chronic stresses--the decline of GKA in period of damage of cell activities, increase of its activity in period of maximum resistance and its following decrease in period of the plant metabolism depletion.
The progamic phase, the period of pollen tube growth through the pistil, is a period of specific interactions between the
male gametophyte and the pistil. Understanding of pollen germination and pollen tube growth are relevant for the study of
pollen-pistil interactions and for understanding the function of components specifically accumulated in the transmitting tissue
cell walls and intercellular matrix that may interact with pollen tubes.
Nicotiana alata has a style-specific hydroxyproline-rich glycoprotein (the 120 kDa glycoprotein) which has properties of both extensins and AGPs [19, 20]. The 120 kDa glycoprotein is a soluble component in the extracellular matrix of the transmitting tract of styles where it accounts for ca. 9% of the total buffer-soluble protein. Here we describe the molecular cloning of a cDNA representing the gene NaPRP5 which encodes the backbone of the 120 kDa glycoprotein. Expression of mRNA is restricted to styles, consistent with observations on the distribution of the 120 kDa glycoprotein. Levels of accumulation of the transcript encoding the 120 kDa protein backbone are not altered significantly by pollination with either compatible or incompatible pollen. The protein backbone of the 120 kDa glycoprotein, as predicted by the cDNA sequence, is composed of three distinct domains. The sequence of these domains, together with linkage analysis of the carbohydrate component of the 120 kDa glycoprotein, allows predictions of the likely distribution of substituent glycosyl chains along the protein backbone. The similarity of the C-terminal domains of the 120 kDa glycoprotein and GaRSGP, the galactose-rich style glycoprotein of N. alata, is consistent with the two molecules sharing a common antigenic domain in their backbones [31]. The sharing of domains between distinct hydroxyproline-rich glycoproteins suggests that identification of a glycoprotein of this class solely by its protein or carbohydrate epitope is not valid.
In higher plants, the sexual organs, e.g. anther and pistil, are separated spatially either within a flower in hermaphrodite plants or in different flowers as in monoecious plants. Successful fertilization requires temporally and spatially coordinated development of male and female organs and gametophytes. Mechanisms have also been evolved to ensure pollen survival in harsh environments and to deliver the male gametes to the female gametophyte enclosed within the ovule, which in turn is embedded in the pistil. Furthermore, crosstalk between the male and female gametophytes ensures the success of fertilization that occurs at high efficiency and in a controlled manner.
The pollen-specific gene Ntp303 belongs to the class of late pollen specific genes. It is first transcribed directly after pollen mitosis. Biochemical properties,
appearance and precise location of the NTP303 protein during pollen development and pollen tube growth were studied by amino-acid
micro-sequencing, protein gel blotting and immuno-localization. Antisera were raised against recombinant proteins, encoded
by sequences of the pollen-specific Ntp303 gene. The antibodies specifically recognized a 69-kDa glycoprotein. Electron-microscopic immuno-localization of the protein
revealed the presence of high concentrations of the NTP303 protein at the vegetative plasma membranes that surround the vegetative
cell, the generative cell and the sperm cells of pollen and pollen tubes. The generative plasma membranes of the generative
cell and the sperm cells were negative. NTP303 protein was also present in the cell walls and in callose plugs. With this
method it was shown that the NTP303 protein was already present in mid-bicellular pollen, after the first, asymmetrical pollen
mitosis. Possible functions for the NTP303 protein are discussed in relation to its properties and its association with the
vegetative plasma membranes.
A new method for the chemical deglycosylation of glycoproteins is described. Treatment of fetuin with trifluoromethanesulfonic acid at 0 or 25°C results in rapid cleavage of peripheral sugars, slow loss of serine- and threonine-linked N-acetylgalactosamine, and retention of N-glycosidically linked N-acetylglucosamine. Amino acid analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveal that the peptide backbone is left intact after reaction at 0°C for 2.5 h or at 25°C for 1 h. Treatment of ovine luteinizing hormone and human M,N-active erythrocyte sialoglycoproteins at 0°C for 3 h gives similar results. In the case of ovine luteinizing hormone, the recovered deglycosylated hormone has approximately 2% of the starting material's activity as measured in a receptor binding assay. In the case of bovine nasal septum proteoglycan, the deglycosylated core protein acts as an excellent acceptor for UDP-d-xylose:core protein β-O-xylosyltransferase. The reagent is far less toxic than HF and does not require special equipment for handling. The procedure should prove useful not only for generation of deglycosylated proteins, but also for assessment of the number of asparagine-linked saccharide chains in a glycoprotein.
We have sought to identify pistil-specific genes that can be used as molecular markers to study pistil development. For this purpose, a cDNA library was constructed from poly(A)+ RNA extracted from tobacco stigmas and styles at different developmental stages. Differential screening of this library led to the isolation of cDNA clones that correspond to genes preferentially or specifically expressed in the pistil. Seven of these cDNA clones encode proteins containing repetitions of the pentapeptide Ser-Pro4, which is a typical motif found in extensins. Unlike extensin genes, the extensin-like genes described here are not induced under stress conditions. RNA gel blot hybridizations demonstrated the organ-specific expression of the extensin-like genes and their temporal regulation during pistil development. After pollination, the transcript levels of the pistil-specific extensin-like genes change relative to levels in unpollinated pistils. In situ hybridization experiments showed that at least one of these pistil-specific genes is specifically expressed in cells of the transmitting tissue. The possible roles of the extensin-like proteins in pistils are discussed.
In plant sexual reproduction, pollen tubes elongate from the stigma, through the stylar transmitting tissue, to the ovary of the pistil to deliver the male gametes for fertilization. TTS protein is a tobacco transmitting tissue glycoprotein shown to attract pollen tubes and promote their growth. Here, we show TTS proteins adhere to the pollen tube surface and tips, suggesting that they may serve as adhesive substrates for pollen tube growth. TTS proteins are also incorporated into pollen tube walls and are deglycosylated by pollen tubes, suggesting that they may provide nutrients to this process. Within the transmitting tissue, TTS proteins display a gradient of increasing glycosylation from the stigmatic end to the ovarian end of the style, coincident with the direction of pollen tube growth. These results together suggest that the TTS protein-bound sugar gradient may contribute to guiding pollen tubes from the stigma to the ovary.
Pollen tubes elongate directionally in the extracellular matrix of pistil tissues to transport the male gametes from the apically located stigma to the basally located ovary for fertilization. The molecular mechanisms underlying directional pollen tube growth in the pistil are poorly understood. We have purified a glycoprotein, TTS, from tobacco stylar transmitting tissue, which supports pollen tube growth between the stigma and the ovary. TTS proteins belong to the arabinogalactan protein family, and they polymerize readily in vitro in a head-to-tail fashion into oligomeric forms. TTS proteins stimulate pollen tube growth in vitro and attract pollen tubes grown in a semi-in vivo culture system. In vivo, the pollen tube growth rate is reduced in transgenic plants that have significantly reduced levels of TTS proteins as a result of either antisense suppression or sense cosuppression. These results identify TTS protein as a pistil component that positively contributes to pollen tube growth.
The pistil of flowers is a specialized organ which contains the female gametophytes and provides the structures necessary for pollination and fertilization. Pollen deposited on the stigmatic surface of a compatible plant germinates a pollen tube which penetrates the stigmatic papillae and grows intercellularly through the style towards the ovules in the ovary. Pollen tube growth is largely restricted to the transmitting tissue in the style. Therefore the stylar transmitting tissue is extremely important for the migration of the pollen cell towards the ovary. We have isolated two related cDNAs, transmitting tissue-specific (TTS)-1 and TTS-2, derived from two proline-rich protein (PRP)-encoding mRNAs that accumulate specifically in the transmitting tissue of tobacco. The deduced PRP sequences share similarities with proline-rich cell wall glycoproteins found in a variety of plants. TTS-1 and TTS-2 mRNAs are induced in very young floral buds, accumulate most abundantly during the later stages of flower development when style elongation is the most rapid, and remain at relatively high levels at anthesis. These mRNAs become undetectable in maturing green fruits. In situ hybridization shows that TTS-1 and TTS-2 mRNA accumulation is restricted to the transmitting tissue of the style. The possible roles that these transmitting tissue-specific PRPs may play in maintaining the structural integrity of the style or in the function of this organ is discussed.
Arabinogalactan-proteins (AGPs) are proteoglycans with a high level of galactose and arabinose. Their current functions in plant development remain speculative. In this study, (beta-D-glucosyl)3 Yariv phenyl-glycoside [(beta-D-Glc)3] was used to perturb AGPs at the plasmalemma-cell wall interface in order to understand their functional significance in cell wall assembly during pollen tube growth. Lily (Lilium longiflorum Thunb.) pollen tubes, in which AGPs are deposited at the tip, were used as a model. Yariv phenylglycoside destabilizes the normal intercalation of new cell wall subunits, while exocytosis of the secretory vesicles still occurs. The accumulated components at the tip are segregated between fibrillar areas of homogalacturonans and translucent domains containing callose and AGPs. We propose that the formation of AGP/(beta-D-Glc)3 complexes is responsible for the lack of proper cell wall assembly. Pectin accumulation and callose synthesis at the tip may also change the molecular architecture of the cell wall and explain the lack of proper cell wall assembly. The data confirm the importance of AGPs in pollen tube growth and emphasize their role in the deposition of cell wall subunits within the previously synthesized cell wall.
In flowering plants, guidance of the pollen tube to the embryo sac (the haploid female gametophyte) is critical for successful
fertilization. The target embryo sac may attract the pollen tube as the final step of guidance in the pistil. We show by laser
cell ablation that two synergid cells adjacent to the egg cell attract the pollen tube. A single synergid cell was sufficient
to generate an attraction signal, and two cells enhanced it. After fertilization, the embryo sac no longer attracts the pollen
tube, despite the persistence of one synergid cell. This cessation of attraction might be involved in blocking polyspermy.
The fertilization process of plants is governed by different kinds of cell-cell interactions. In higher plants, these interactions are required both for recognition of the pollen grain by the female reproductive system and to direct the growth of the pollen tube inside the ovary. Despite many years of study, the signaling mechanisms that guide the pollen tube toward its target, the ovule, are largely unknown. Two distinct types of principles, mechanical and chemotropic, have been suggested to account for the directed growth of the pollen tube. The first of these two types of models implies that the guidance of the pollen tube depends on the architecture and chemical properties of the female reproductive tissues, whereas the latter suggests that the ovule provides a signal for the target-directed growth of the pollen tube. To examine such a role for the ovules, we analyzed the growth path of pollen tubes in mutants defective in ovule development in Arabidopsis. The results presented here provide unique in vivo evidence for an ovule-derived, long-range activity controlling pollen tube guidance. A morphological comparison of the ovule mutants used in this study indicates that within the ovule, the haploid embryo sac plays an important role in this long-range signaling process.
The class III pistil‐specific PELP proteins (PELPIII) of Nicotiana tabacum includes at least two members of highly soluble glycoproteins containing glucan modules that are characteristic for arabinogalactan
proteins (AGPs). PELPIII accumulates in the style transmitting tissue (TT) during pistil development and, at flower anthesis,
is present in the intercellular matrix (IM) of non‐pollinated pistils. After pollination, PELPIII appears to be directly and
completely translocated from the IM into the pollen tube callose walls, no significant accumulation was observed in the primary
wall in the tip. In the spent parts of the pollen tubes these proteins become detectable against the remnants of the tube
cell membrane and in the callose plugs. Different protein extraction procedures of PELPIII from pollinated tobacco pistils
showed that these proteins remain in the highly soluble protein fraction and are not modified by the growing pollen tubes.
These data concur with a role in IM development and pollen tube growth. In addition, the data show that the PELPIII are able
to reach the cell membrane, facilitated by an already present or induced high porosity of the tube wall and an additional,
yet unknown, mechanism. The differences in behaviour between the three related classes of style IM glycoproteins of Nicotiana, namely, PELPII, TTS and the120 kDa glycoprotein, are proposed to connect more to their differences in glycosylation than
to major differences in amino acid sequence.
Many aspects of Angiosperm pollen germination and tube growth are discussed including mechanisms of dehydration and rehydration, in vitro germination, pollen coat compounds, the dynamic involvement of cytoskeletal elements (actin, microtubules), calcium ion fluxes, extracellular matrix elements (stylar arabinogalactan proteins), and control mechanisms of gene expression in dehydrating and germinating pollen. We focus on the recent developments in pollen biology that help us understand how the male gamete survives and accomplishes its successful delivery to the ovule of the sperm to effect sexual reproduction.
This chapter discusses the role of exudate as a source of carbohydrate precursors for pollen tube wall formation. Labeling experiments with Lilium longiflorum have demonstrated that carbohydrate material derived from germinating medium or pistil is used by pollen during tube wall formation. For these experiments, the flowers of Easter Lily, Lilium longiflorum from plants grown under greenhouse conditions were used. Although the chromatographic separation used in this study did not fully resolve labeled galactose and labeled glucose, it was possible to show that fractionation of the exudate removed almost all labeled glucose from the high molecular weight fraction. Results obtained from the in vitro and in situ experiments indicate that growing pollen tubes use pistil exudate for tube wall biosynthesis.
The presence of presynthesized messenger RNAs in the mature, dehydrated pollen grains of Tradescantia paludosa L. has been demonstrated by translation of total RNA and poly (A) + RNA in a wheat germ cell-free system, and a comparison of in vitro and in vivo synthesized proteins by SDS-polyacrylamide gel electrophoresis. The mRNAs are capped at their 5'-termini with a guanosine 5' phosphate moiety which is methylated.
It has been proposed that a stylar glycoprotein, the transmitting tissue-specific (TTS) protein isolated from Nicotiana tabacum, serves both as a growth stimulant, by providing a source of nutrients, and as an attractant for pollen tubes during their growth through the style. Working with a galactose-rich style glycoprotein (GaRSGP) that is the N. alata homologue (97% homology) of the TTS protein, a series of experiments, similar to those done with the TTS protein, was performed. Evidence was found for inhibition of pollen tube growth at high concentrations, but no evidence was found for stimulation of growth at concentrations up to 2 mg ml–1. No effect as a pollen tube attractant was detected. The discrepancies in the features and functionality between these homologous glycoproteins in the closely related Nicotiana species warrants further investigation before a general function is assigned to these molecules.
There appears to be interest, also on the part of private breeders, in studying pollen tubes with the aid of fluorescence.The method described, which is applied in research on self- and cross-incompatibility, makes a rapid and correct interpretation possible.
This review summarises information on hydroxyproline-rich glycoproteins (HRGPs) and clones encoding these molecules isolated
from male and female tissues of flowering plants. HRGPs are abundant in sexual tissues and a number of different HRGPs have
been isolated. The protein and carbohydrate components of these HRGPs have been characterised and cDNAs encoding the protein
backbones of several have been isolated and sequenced. Further work is directed at detailed structural analysis of the carbohydrate
side chains of these molecules and their points of linkage to the protein backbones. The biological functions of most of these
molecules have not yet been established unequivocally.
Growth of pollen tubes ofNicotiana tabacum W 38 in a defined liquid medium buffered at pH 5.9 and containing sucrose, amino-acids, boric acid, salts and an antibacterial agent was stimulated by the addition of poly(ethylene glycol) 6000 (PEG-6000) and Cu(II) salts. In the absence of both these supplements, up to 50% of the hydrated pollen grains did not develop further, and the germinated tubes were slow-growing and abnormal, with thickened walls, kinked growth, and fragile, swollen tips containing granular cytoplasm. Addition of 10–15% (w/v) purified PEG-6000 increased germination to 80–90% and prevented the progressive bursting of pollen grains and tube tips, but growth was still slow and kinked and tips remained swollen. Addition of 30 M CuSO4 did not stimulate germination or prevent tip bursting, but produced straight-growing tubes with smooth-sided tips resembling the tips of tubes growing through stylar tissue; the free Cu2+ concentration under these conditions was about 1.0 M due to chelation by amino-acids, and similar tube morphologies were obtained with 1.0–1.5 M added CuSO4 when NH4Cl replaced the amino-acids. When the medium containing amino-acids was supplemented with both 12.5% PEG-6000 and 30 M CuSO4, long-term (48 h) growth of straight pollen tubes with smooth-sided tips, thin walls and long ladders of callose plugs was observed; growth occurred at 250 m/h, approximately 30–40% of the rate observed in the style. Although omission of CuSO4 from this complete medium severely affected tube growth and callose plug deposition, it did not alter the timing of generative-nucleus division, and thus the different parameters associated with the second phase of pollen-tube growth can be uncoupled in culture. High levels of FeSO4 (300 M) had a similar morphogenetic effect to CuSO4, but addition of 300 M L-ascorbate or D-iso-ascorbate was required to prevent precipitation of Fe(III) oxide and prolong the stimulation of pollen-tube growth; EDTA removed the morphogenetic effect of both CuSO4 and FeSO4. Further, an impure grade of PEG-4000 was contaminated with an organic morphogen that allowed continued slow growth of pollen tubes with smooth, straight-sided tips in the absence of added CuSO4 or FeSO4, with tube morphology unaffected by ascorbate or EDTA. However, the long-term morphogenetic effect of trace levels of CuSO4 suggests that Cu(II) salts play an important role in pollen-tube development in at least this species ofNicotiana.
Arabinogalactan proteins (AGPs) are a class of plant extracellular-matrix proteins believed to participate in a broad range of processes involving the plant cell surface. They are extremely abundant in female reproductive tissues and in pollen tubes, the haploid male structures that traverse the diploid female reproductive tissues to deliver sperms to the egg cells. The prevalence of AGPs in reproductive tissues has led to speculations that they play significant functional roles ranging from serving as nutrient resources to cell-cell recognition in plant reproduction. Recent research from several laboratories demonstrated functional participation by AGPs in reproductive processes and began to examine the mechanisms underlying these functional roles. An overview of these recent studies will be discussed with a historical perspective as well as with a view towards future studies in establishing the significance of AGPs that, as a class, they have prominent roles in plant sexual reproduction in multiple and diverse ways.
Nicotiana tabacum was used as a pistillate parent and crossed with three self-compatible species, N. rustica, N. repanda and N. trigonophylla, which were previously reported to have pollen tubes unilaterally inhibited by N. tabacum pistil. Temporal and morphological observations revealed distinct differences of pollen tube behavior among these incongruous crosses. Pollen tubes of N. repanda were arrested in stigma and those of N. rustica in the middle of the style. On the other hand, pollen tubes of N. trigonophylla continued growing at a slow rate. Tubes of N. repanda and N. rustica showed morphological abnormalities such as swelling, thick wall, and irregular callose deposition. In addition, tubes of N. rustica often elongated in reverse direction and wound about in the middle of the style. Although the tubes of N. trigonophylla were apparently normal in morphology, they were distributed throughout the transmitting tissue, differing from the self-pollination of N. tabacum in which they were confined to the peripheral region of it. The diversity of pollen tube behavior indicates that physiological causes of incongruity are different among the three crosses. Bud pollination enabled pollen tubes to reach the ovary in all crosses, indicating that the N. tabacum pistil acquired its ability to inhibit foreign pollen tube elongation with its development. When interspecific hybrids between N. tabacum and the other three species were pollinated by parental species, tubes reached the ovary in all crosses, but the elongation rate of tubes slowed down and morphology was abnormal.
Inter- and intra-specific compatibility systems in the genus Nicotiana have been used to identify factors that control pollen recognition. N. alata has a classic gametophytic self-incompatibility (SI) system in which the specificity of pollen rejection is determined by a multiallelic S-locus. S-RNases are products of the S-locus, and are the factors that determine specificity in the style. In vitro mutagenesis experiments have been conducted to determine how allelic specificity is encoded in the S-RNase sequence. Plant transformation experiments have shown that S-RNases also act as factors controlling interspecific pollen rejection. By examining the effect of S-RNases on inter- and intra-specific compatibility in different genetic backgrounds, four different pollen rejection mechanisms can be recognized. S-RNases are implicated in three of these mechanisms. The dependence of pollen rejection on genetic background shows that S-RNases interact with other factors. In general, such factors can be classified in three groups based on their mode of interaction with the S-locus and other pollen-pistil interaction pathways. Some of these factors are now cloned. As more factors are cloned and characterized, it is becoming apparent that pollen-pistil interactions that were once thought to be distinct are actually interrelated.
The major component of the Gladiolus style mucilage was shown to be an arabinogalactan-protein. The arabinogalactan-protein was isolated from the style extract by affinity chromatography with tridacnin (the galactose-binding lectin from the clam Tridacna maxima) coupled to Sepharose 4B. The isolated arabinogalactan-protein represents 40% of the soluble style extract; it contains 90% (w/w) carbohydrate and 3% protein. The major monosaccharides of the carbohydrate component are galactose and arabinose, in the proportions 6:1. A component with a similar composition was also isolated from the crude extract by precipitation with the beta-glucosyl artifical carbohydrate antigen. The protein moiety of the arabinogalactan-protein remained associated with the carbohydrate after chromatography in urea, and has high contents of serine, glutamic acid, aspartic acid, glycine and alanine. The arabinogalactan-protein is apparently chemically homogeneous; it eluted as a single symmetrical peak from Sepharose 4B, and three fractions collected across the peak were structurally similar. Ultracentrifugal studies showed it to be polydisperse in the mol.wt. range 150 000--400 000. The information obtained from methylation analyses, oxalic acid and enzymic hydrolyses is consistent with a model having a beta 1 leads to 3 galactan backbone, branched through C(O)6 to beta 1 leads to 6 galactan side chains. The arabinose is exclusively present as terminal alpha-L-arabinofuranosyl residues. Enzymic removal of the arabinose residues resulted in a marked decrease in solubility of the molecule. The localization of the arabinogalactan-protein in the mucilage of the style canal was demonstrated cytochemically. The possible roles of the arabinogalactan-protein in relation to recognition of compatible pollen and pollen-tube growth are discussed.
A protein determination method which involves the binding of Coomassie Brilliant Blue G-250 to protein is described. The binding of the dye to protein causes a shift in the absorption maximum of the dye from 465 to 595 nm, and it is the increase in absorption at 595 nm which is monitored. This assay is very reproducible and rapid with the dye binding process virtually complete in approximately 2 min with good color stability for 1 hr. There is little or no interference from cations such as sodium or potassium nor from carbohydrates such as sucrose. A small amount of color is developed in the presence of strongly alkaline buffering agents, but the assay may be run accurately by the use of proper buffer controls. The only components found to give excessive interfering color in the assay are relatively large amounts of detergents such as sodium dodecyl sulfate, Triton X-100, and commercial glassware detergents. Interference by small amounts of detergent may be eliminated by the use of proper controls.
This report describes the isolation and characterization of a cDNA clone representing a gene specifically expressed in pollen. A cDNA library was constructed against mRNA from mature pollen of Nicotiana tabacum. It was screened differentially against cDNA from mRNA of leaf and of pollen. One clone, NTPc303, was further characterized. On northern blot this clone hybridizes to a transcript 2100 nucleotides in length. NTPc303 is abundant in pollen. Expression of the corresponding gene is restricted to pollen, because no other generative or vegetative tissue contains transcripts hybridizing to NTPc303. Expression of NTP303 is evolutionarily conserved: homologous transcripts are present in pollen from various plant species. The first NTP303 transcripts are detectable on northern blot at the early bi-nucleate stage and accumulate until the pollen has reached maturity. During germination and pollen tube growth in vitro new NTP303 transcripts appear. This transcription has been proved by northern blots as well as by pulse labelling experiments. Nucleotide sequence analysis revealed that NTPc303 has an open reading frame coding for a predicted protein of 62 kDa. This protein shares homology to ascorbate oxidase and other members of the blue copper oxidase family. A possible function for this clone during pollen germination is discussed.
An approach for performing multiple alignments of large numbers of amino acid or nucleotide sequences is described. The method is based on first deriving a phylogenetic tree from a matrix of all pairwise sequence similarity scores, obtained using a fast pairwise alignment algorithm. Then the multiple alignment is achieved from a series of pairwise alignments of clusters of sequences, following the order of branching in the tree. The method is sufficiently fast and economical with memory to be easily implemented on a microcomputer, and yet the results obtained are comparable to those from packages requiring mainframe computer facilities.
Plants represent a diverse group of organisms that have unique reproductive, developmental, and physiological processes. Although
morphologically simple, plants have molecular genetic processes that are equivalent in complexity to those found in animals.
Sophisticated gene transfer procedures, transposon mutagenesis in homologous and heterologous plants, and development of model
organisms such as Arabidopsis permit almost any gene that is associated with an observable phenotype to be isolated and studied.
These advances, coupled with general advances in molecular biology, now make it possible to dissect the molecular and cellular
events responsible for controlling plant-specific processes.
Soybean embryo mRNA populations were compared with respect to complexity, abundance distribution, and sequence representation. RNA-excess hybridization experiments with cDNA and single-copy DNA showed that cotyledon-stage (30 days after flowering (DAF)) and mid-maturation-stage (75 DAF) embryos contained similar numbers of diverse mRNAs (14,000–18,000) but that their mRNA frequency distributions were vastly different. Hybridization experiments with kinetically selected mid-maturation-stage abundant and rare cDNAs demonstrated that the diverse mRNAs of cotyledon-stage and mid-maturation-stage embryos were similar, and that most embryonic mRNAs persist throughout development and are stored in the dormant seed. We have, however, identified a small number (7–10) of superabundant mRNAs which collectively comprise about 50% of the mid-maturation-stage mRNA mass. These mRNAs are quantitatively modulated in development, are embryo specific, and are encoded by low-frequency repetitive DNA sequences. This developmentally regulated mRNA set probably codes for a spectrum of seed proteins which accumulate during maturation.
A basic, hydroxyproline-rich glycoprotein (molecular mass 120 kDa) has been purified from the styles of Nicotiana alata. An antibody, specific for the protein backbone (molecular mass 78 kDa) of the glycoprotein, was used to demonstrate that the glycoprotein is a soluble, style-specific component and that related molecules are present in the styles of other solanaceous species. Linkage analysis of the carbohydrate portion of the glycoprotein, together with antibody binding studies, indicates that the glycoprotein contains both extensin-like and arabinogalactan-protein (AGP)-like side chains. Furthermore, the AGP-like side-chains contain a style-specific epitope that is also present on AGPs from N. alata styles and glycoconjugates from the styles of other members of the Solanaceae. The abundance of this 120 kDa glycoprotein, its location in the extracellular matrix of the transmitting tract and its conservation in several species within the Solanaceae suggests a role in pistil function.
cDNA clones encoding a novel proline-rich protein (NaPRP4) have been isolated from a Nicotiana alata stylar cDNA library. The N-terminal part of the derived protein is highly rich in proline (32.2%) and contains several repeats such as Lys-Pro-Pro (7 times) and Pro-Thr-Lys-Pro-Pro-Thr-Tyr-Ser-Pro-Ser-Lys-Pro-Pro (twice); the C-terminal part, on the other hand, has a lower proline content (9.9%) and contains two potential N-glycosylation sites and all the six cysteine residues. Northern blot and in situ hybridisation analyses indicate that expression of the NaPRP4 gene is restricted to cells of the transmitting tract of the style.
A basic, galactose-rich style glycoprotein (GaRSGP) encoded by a previously characterized style-specific cDNA (NaPRP4) has been isolated from the styles of Nicotiana alata and structurally characterized. The glycoprotein is associated with cell walls in the transmitting tract and is composed of approximately 25% (w/w) protein and 75% (w/w) carbohydrate. The purified glycoprotein appears as a smear of between 45-120 kDa on SDS-PAGE; the deglycosylated protein backbone has an apparent molecular weight of approximately 30 kDa. The glycoprotein is rich in the amino acids lysine, proline, and hydroxyproline and in the monosaccharides galactose and arabinose. It is one of only a few proline/hydroxyproline-rich glycoproteins (P/HRGPs) to be characterized both as a cDNA-clone and protein. Glycans are attached to the protein backbone through both O- and N-glycosidic linkages with the majority of the carbohydrate being O-linked and consisting of short, highly branched chains terminating primarily in galactose residues. A carbohydrate epitope(s) is found on both GaRSGP and another style-specific glycoprotein but not on glycoproteins from other tissues. This finding provides further evidence for the existence of a style-specific carbohydrate epitope(s) which may play a role in style function.
Transmitting tissue-specific (TTS) protein is a pollen tube growth-promoting and attracting glycoprotein located in the stylar transmitting tissue extracellular matrix of the pistil of tobacco. The TTS protein backbones have a deduced molecular mass of about 28 kDa, whereas the glycosylated stylar TTS proteins have apparent molecular masses ranging between 50 and 100 kDa. TTS mRNAs and proteins are ectopically produced in transgenic tobacco plants that express either a cauliflower mosaic virus (CaMV) 35S promoter-TTS2 transgene or a CaMV 35S-promoter-NAG1 (NAG1 = Nicotiana tabacum Agamous gene) transgene. However, the patterns of TTS mRNA and protein accumulation and the quality of the TTS proteins produced are different in these two types of transgenic plants. In 35S-TTS transgenic plants, TTS mRNAs and proteins accumulate constitutively in vegetative and floral tissues. However, the ectopically expressed TTS proteins in these transgenic plants accumulate as underglycosylated protein species with apparent molecular masses between 30 and 50 kDa. This indicates that the capacity to produce highly glycosylated TTS proteins is restricted to the stylar transmitting tissue. In 35S-NAG transgenic plants, NAG1 mRNAs accumulate constitutively in vegetative and floral tissues, and TTS mRNAs are induced in the sepals of these plants. Moreover, highly glycosylated TTS proteins in the 50- to 100-kDa molecular mass range accumulate in the sepals of these transgenic, 35S-NAG plants. These results show that the tobacco NAGI gene, together with other yet unidentified regulatory factors, control the expression of TTS genes and the cellular capacity to glycosylate TTS proteins, which are normally expressed very late in the pistil developmental pathway and function in the final stage of floral development. The sepals in the transgenic 35S-NAG plants also support efficient pollen germination and tube growth, similar to what normally occurs in the pistil, and this ability correlates with the accumulation of the highest levels of the 50- to 100-kDa glycosylated TTS proteins.
Pollination induces many physiological responses in the flower, including deterioration and death in specific pistil cell types. It is shown here that within the style of tobacco, pollination-induced cell deterioration was restricted to the transmitting tissue while the surrounding cortical tissue was not affected. It was distinct from general senescence since exogenously applying the senescence-inducing hormone ethylene, or its precursor aminocyclopropane-1-carboxylic acid (ACC), to the flower or the pistil induced overall deterioration in the entire flower. Furthermore, both pollen tube growth and ethylene action were needed for the entire spectrum of cellular changes associated with this pollination-induced transmitting tissue deterioration process. It is also shown that pollination-induced mRNA poly(A) tail-shortening for at least three major classes of transmitting tissue-specific mRNAs. As is commonly observed for poly(A) tail-shortened mRNAs, the levels of two of these three mRNA classes decline after pollination. On the other hand, the third class of mRNAs, transmitting tissue-specific (TTS) mRNAs, was maintained at a very high level subsequent to pollination, even after substantial poly(A)-tail shortening. TTS mRNAs encode a pollen tube growth-promoting and -attracting protein needed for optimal in vivo pollen tube growth. The specific preservation of TTS mRNAs in the deteriorating transmitting tissue cells suggests that these cells can distinguish molecules needed in the pollinated styles from those that are dispensable, and protect them from degradation. It is suggested that the pollination-induced mRNA poly(A) tail-shortening and cell death are programmed processes suited to the post-pollination transmitting tissue environment. Results showing that ACC is a candidate signal molecule for the pollination-induced mRNA-shortening which is accentuated by ethylene and mediated via a protein phosphorylation-dependent signal transduction pathway are also presented.
Upon germination on the stigma, pollen tubes elongate in the stylar transmitting tract, aided by female factors, with speed and directionality not mimicked in in vitro pollen tube growth cultures. We have shown that a stylar transmitting tissue arabinogalactan protein (AGP) from Nicotiana tabacum (tobacco), TTS protein, stimulates pollen tube growth in vivo and in vitro and attracts pollen tubes grown in a semi-in vivo culture system. It has been reported that the self-incompatible Nicotiana alata produced a stylar glycoprotein, GaRSGP, which had a backbone polypeptide that shared 97% identity with those of TTS proteins but some of its properties were different from those described for TTS proteins. We report here the characterization of a family of stylar transmitting tissue glycoproteins from N. alata that is virtually identical to tobacco TTS proteins and which we refer to as NaTTS proteins. Like their tobacco counterparts, NaTTS proteins are recognized by the traditional AGP-diagnostic reagent beta-glucosyl Yariv reagent, and they are also recognized by JIM13, a monoclonal antibody against AGP. NaTTS proteins also stimulate pollen tube elongation in vitro and attract pollen tubes in a semi-in vivo pollen tube culture system. Biochemical and immunological characterization of NaTTS proteins revealed that they have extraordinary variability in the extent of sugar modifications of their polypeptide backbones. The extent of sugar modifications on NaTTS proteins significantly affects their biochemical properties, influences how they interact with the transmitting tissue extracellular matrix, and affects their solubility from this matrix. Our results suggest that the strategy used to purify GaRSGP only recovered a less glycosylated, more tightly extracellular matrix-bound sub-population of the entire spectrum of N. alata TTS proteins.
Sexual reproduction in plants, unlike that of animals, requires the action of multicellular haploid gametophytes. The male gametophyte (pollen tube) is guided to a female gametophyte through diploid sporophytic cells in the pistil. While interactions between the pollen tube and diploid cells have been described, little is known about the intercellular recognition systems between the pollen tube and the female gametophyte. In particular, the mechanisms that enable only one pollen tube to interact with each female gametophyte, thereby preventing polysperm, are not understood. We isolated female gametophyte mutants named magatama (maa) from Arabidopsis thaliana by screening for siliques containing half the normal number of mature seeds. In maa1 and maa3 mutants, in which the development of the female gametophyte was delayed, pollen tube guidance was affected. Pollen tubes were directed to mutant female gametophytes, but they lost their way just before entering the micropyle and elongated in random directions. Moreover, the mutant female gametophytes attracted two pollen tubes at a high frequency. To explain the interaction between gametophytes, we propose a monogamy model in which a female gametophyte emits two attractants and prevents polyspermy. This prevention process by the female gametophyte could increase a plant's inclusive fitness by facilitating the fertilization of sibling female gametophytes. In addition, repulsion between pollen tubes might help prevent polyspermy. The reproductive isolations observed in interspecific crosses in Brassicaceae are also consistent with the monogamy model.
Class III pistil-specific extensin-like proteins (PELPIII) are specifically localized in the intercellular matrix of tobacco (Nicotiana tabacum) styles. After pollination the majority of PELPIII are translocated into the callosic layer and the callose plugs of the pollen tubes, which could suggest a function of PELPIII in pollen tube growth. PELPIII may represent one of the chemical and/or physical factors from the female sporophytic tissue that contributes to the difference between in vivo and in vitro pollen tube growth. PELPIII glycoproteins were purified and biochemically characterized. Because of their high proline (Pro) and hydroxy-Pro (Hyp) content, PELPIII proteins belong to the class of Pro/Hyp-rich glycoproteins. The carbohydrate moiety of PELPIII is attached through O-glycosidic linkages and comprises more than one-half the total glycoprotein. Deglycosylation of PELPIII revealed two backbones, both reacting with PELPIII-specific antibodies. N-terminal amino acid sequencing of these backbones showed that PELPIII is encoded by the MG14 and MG15 genes. Two heterogeneous N-terminal sequences of MG14 and MG15, both starting downstream of the predicted signal peptide cleavage site, seem to be present, which indicates a novel N-terminal processing. Monosaccharide analysis showed that the carbohydrate moiety of PELPIII almost completely consists of arabinose and galactose in an equal molar ratio. Carbohydrate linkage analysis showed terminal and 2-linked arabinofuranosyl residues, as well as terminal and 6-, 3-, and 3,6-linked galactopyranosyl residues to be present, indicating the presence of both extensin-like and Type II arabinogalactan oligosaccharide units. The ability of beta-glucosyl Yariv reagent to bind with PELPIII confirmed the arabinogalactan protein-like characteristics of these proteins.
Arabinogalactan proteins (AGPs) are secreted or membrane-associated glycoproteins that have been operationally defined as binding to [beta]-glucosyl Yariv artificial antigen, being rich in arabinose and galactose, and containing high levels of alanine, serine, and hydroxyproline. Using an anti-AGP monoclonal antibody (MAC 207) bound to cyanogen bromide-activated Sepharose 4B, we have purified by immunoaffinity chromatography an extracellular AGP from the culture medium of suspension-cultured cells of carrot (Daucus carota). The apparent molecular mass of this highly glycosylated proteoglycan is 70 to 100 kD as judged by sodium dodecyl sulfate-polyacrylamide gels. Although its sugar analysis, [beta]-glucosyl Yariv binding, and high alanine, serine, and proline content are consistent with it being an AGP, the amino acid composition unexpectedly revealed this molecule to have no detectable hydroxyproline. This suggests that this glycoprotein is not a "classical" AGP, but represents the first example of a new class of hydroxyproline-poor AGPs. Deglycosylation of the AGP with anhydrous hydrogen fluoride revealed that the purified proteoglycan contains probably a single core protein with an apparent molecular mass of 30 kD. Direct visualization of the native AGP in the electron microscope showed ellipsoidal putative AGP monomers, approximately 25 nm by 15 nm, that showed a strong tendency to self assemble into higher-order structures. Upon desiccation, the glycosylated AGP formed paracrystalline arrays visible in the light microscope. Polarized Fourier transform infrared microspectroscopy of these arrays demonstrated a high degree of polarization of the sugar moieties under these conditions. These results put possible constraints on current models of AGP structure; a putative role for these novel AGPs as pectin-binding proteins is discussed.
The extracellular matrix of stylar transmitting tissues of many angiosperms is enriched in secretory materials that are believed to be important for interactions with pollen tubes. We have previously characterized two related cDNAs (TTS-1 and TTS-2) for stylar transmitting tissue-specific proline-rich proteins (TTS proteins) from Nicotiana tabacum. We show here that TTS proteins are highly glycosylated proteins with apparent molecular masses ranging between 50 and 100 kD. Results from chemical and enzymatic deglycosylation suggest that TTS proteins have N-linked glycosyl groups, and the extensive glycosylation most probably has resulted from modifications at the proline residues. TTS proteins are localized to the intercellular regions between neighboring transmitting tissue cells, the space in which pollen tubes elongate as they migrate from the stigma toward the ovary. TTS mRNA and protein levels are regulated during pistil development and by pollination. The levels of TTS mRNAs and proteins increase with flower development and reach the maximal levels as flowers approach anthesis. These maximal levels are maintained in the styles for at least 3 to 4 days after pollination, during which time pollen tubes elongate and reach the ovary. Spatially, TTS mRNAs and proteins accumulate first in the stigmatic end of young styles, and their levels progressively increase toward the basal end as pistils mature. Pollination stimulates the levels of TTS mRNAs and proteins in hand-pollinated young styles, which normally accumulate relatively low levels of these TTS gene products. Pollination also qualitatively affects TTS mRNAs and proteins. In pollinated styles, TTS mRNAs are shorter than those in unpollinated styles and underglycosylated TTS protein species begin to accumulate. The elaborate regulatory mechanisms governing TTS mRNAs and proteins during development and by pollination strongly suggest that these proteins may play a functional role in the process of pollination.
' The extremely reduced, three-celled, haploid male plant (male gametophyte) of flowering plants has a number of specialized functions to perform. The primary functions are the produc- tion of two sperm cells and their transport within the pollen tube through the tissues of the style and ovary into the em- bryo sac in the ovule, where they participate in double fertilization. The early part of the life of the male gametophyte, following meiosis, occurs within the sporophytic tissues of the anther. During this period, a number of critical developmental events occur that are preceded or accompanied by the induc- tion of activity of a large number of genes (for reviews, see Mascarenhas, 1990,1992; McCormick, 1991; Scott et al., 1991; Bedinger, 1992). The molecular events that take place during
We have identified and characterized the temporal and spatial regulation of a plasma membrane arabinogalactan protein epitope during development of the aerial parts of oilseed rape using the monoclonal antibody JIM8. The JIM8 epitope is expressed by the first cells of the embryo and by certain cells in the sexual organs of flowers. During embryogenesis, the JIM8 epitope ceases to be expressed by the embryo proper but is still found in the suspensor. During differentiation of the stamens and carpels, expression of the JIM8 epitope progresses from one cell type to another, ultimately specifying the endothecium and sperm cells, the nucellar epidermis, synergid cells, and the egg cell. This complex temporal sequence demonstrates rapid turnover of the JIM8 epitope. There is no direct evidence for any cell-inductive process in plant development. However, if cell-cell interactions exist in plants and participate in flower development, the JIM8 epitope may be a marker for one set of them.
Structural studies on the arabinogalactan-protein from the stylar canal of Gladiolus gandavensis
- Pa Gleeson
- Clarke