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PubMed1991

Molecular analysis of gene regulation and function during male gametophyte development.

We have characterised three pollen-expressed genes (LAT52, LAT56 and LAT59) from tomato in order to determine their role in pollen development, and to determine the DNA sequences responsible for gene expression in pollen. LAT52 encodes a protein that shows amino acid sequence similarity to a protein encoded by a pollen-specific cDNA clone (pZmc13) isolated from maize, and both proteins have amino acid sequence similarity to Kunitz trypsin inhibitors of soybean and winged bean. The proteins encoded by LAT56 and LAT59 genes are 54% identical at the amino acid level, and show significant sequence similarity to bacterial pectate lyases and to a fungal pectin lyase. Additionally, regions of LAT56 and LAT59 show significant sequence similarity to tryptic peptides of ragweed and Japanese cedar pollen allergens. Preliminary results suggest that plants harboring antisense constructs of the LAT52 coding region show defects in pollen germination and fertilisation; no obvious phenotype was seen in plants harboring antisense constructs of the LAT59 coding region. Promoter fragments of these three LAT genes were fused to the reporter gene GUS and assayed using both a transient system and stably transformed plants. We have identified relatively short regions of the LAT promoters that are important for pollen expression, and are attempting to isolate trans-acting factors that interact with these cis-acting sequences, using both molecular and classical genetic approaches.

Amino Acid Sequence↗
PubMed1995

Promoters from kin1 and cor6.6, two Arabidopsis thaliana low-temperature- and ABA-inducible genes, direct strong beta-glucuronidase expression in guard cells, pollen and young developing seeds.

The ability of most higher plants to withstand freezing can be enhanced by cold acclimation, although the freezing tolerance of plant tissues is also affected by their developmental stage. In addition, low temperature has pleiotropic effects on many plant developmental processes such as vernalization. The interaction between plant development and low temperature implies that some genes are regulated by both environmental factors and developmental cues. Although a number of cold-inducible genes from plants have been identified, information concerning their regulation during plant development is limited. In order to understand their developmental regulation and obtain possible clues as to function, the promoters of kin1 and cor6.6, two cold- and abscisic acid (ABA)-regulated genes from Arabidopsis thaliana, were fused to the beta-glucuronidase (GUS)-coding sequence and the resulting constructs were used to transform tobacco and A. thaliana. Transgenic plants with either the kin1 or cor6.6 promoter showed strong GUS expression in pollen, developing seeds, trichomes and, most interestingly, in guard cells. During pollen development, maximum GUS activity was found in mature pollen. In contrast, the maximum GUS activity during seed development was during early embryogenesis. These patterns of expression distinguish kin1 and cor6.6 from related lea genes which are strongly expressed during late embryogenesis. There was no major qualitative difference in patterns of GUS expression between kin1 and cor6.6 promoters and the results were similar for transgenic tobacco and Arabidopsis. Considering the results described, as well as those in an accompanying paper (Wang et al., 1995, Plant Mol Biol 28: 605-617 (this issue), we suggest that osmotic potential might be a major factor in regulating the expression of kin1 and cor6.6 during several developmental processes. The implication of the results for possible function of the gene products is discussed.

Abscisic Acid↗
PubMed2005

Flavonoids: from cell cycle regulation to biotechnology.

Flavonoids have been proposed to play diverse roles in plant growth and development, including defense, symbiosis, pollen development and male fertility, polar auxin transport, and protection against ultraviolet radiation. Recently, a new role in cell cycle regulation has emerged. Genetic alteration of glucuronide metabolism by altered expression of a Pisum sativum UDP-glucuronosyltransferase (PsUGT1) results in an altered cell cycle in pea, alfalfa, and Arabidopsis. In alfalfa, altered expression of PsUGT1 results in accumulation of a flavonoid-like compound that suppresses growth of cultured cells. The results are consistent with the hypothesis that PsUGT1 functions by controlling cellular levels of a factor controlling cell cycle (FCC).

Biotechnology↗
PubMed1999

Pleiotropic effects of a nuclear restorer-of-fertility locus on mitochondrial transcripts in male-fertile and S male-sterile maize.

Cytoplasmic male sterility (CMS) is encoded by the plant mitochondrial genome and can be reversed by nuclear restorer-of-fertility(Rf) alleles. In the CMS-S system of maize, reproductive failure and fertility restoration are gametophytic, occurring during the starch-filling stages of pollen development. Transcripts of the CMS-S-associated mitochondrial open reading frames (orf355 and orf77) are present from the early stages of microspore development through the aborted pollen stage. To investigate the molecular basis of fertility restoration, we compared mitochondrial-transcript accumulation in aborting CMS-S pollen and in CMS-S pollen restored to fertility by the Rf3 nuclear allele. In the presence of the Rf3 allele, novel, shorter transcripts of the orf355-orf77, cob and atp6 mitochondrial genes were created, and the relative abundance of larger transcripts was decreased for each of these loci. The altered transcript patterns cosegregated with male fertility conditioned by the Rf3 allele. The novel cob and atp6 transcripts were also observed in leaf-tissues of both normal and S-cytoplasm plants carrying the Rf3 allele. These observations support the hypothesis that the Rf3 allele encodes, or regulates, a modifier of mitochondrial transcript (Mmt) activity that affects both CMS and essential mitochondrial gene transcripts.

Alleles↗
PubMed2004

AtSTP11, a pollen tube-specific monosaccharide transporter in Arabidopsis.

Pollen development, as well as pollen germination and pollen tube growth, requires a highly regulated supply of sugars. In this paper we describe the molecular, kinetic, and physiological characterization of AtSTP11, a new member of the H+/monosaccharide transporter family in Arabidopsis thaliana (L.) Heynh. Heterologous expression in yeast (Saccharomyces cerevisiae) showed that AtSTP11 is a high-affinity (Km = 25 microM), broad-spectrum, and uncoupler-sensitive monosaccharide transporter of the plasma membrane. In reverse transcription-polymerase chain reaction analyses we found that AtSTP11 expression is restricted to flowers. Furthermore, AtSTP11-promoter::GFP plants revealed that AtSTP11 expression is only found in pollen tubes. Using a specific antibody we could also detect the AtSTP11 protein exclusively in pollen tubes but not in other flower tissues or in pollen grains of any developmental stage. These results suggest that the newly identified AtSTP11 transporter plays a role in the supply of monosaccharides to growing pollen tubes.

Amino Acid Sequence↗
PubMed2006

A pollen-specific polygalacturonase from lily is related to major grass pollen allergens.

A pollen-specific gene from lily (Lilium longiflorum Thunb. cv. Snow Queen), designated LLP-PG, was characterized. Southern blots of lily genomic DNA indicated that LLP-PG is a member of a small gene family. A thorough sequence analysis revealed that the LLP-PG gene is interrupted by two introns and encodes a protein of 413 amino acids, with a calculated molecular mass of 44 kDa, and a pI of 8.1. Evaluation of the hydropathy profile showed that the protein has a hydrophobic segment at the N-terminus, indicating the presence of a putative signal peptide. A sequence similarity search showed a significant homology of the encoded protein to pollen polygalacturonases (PGs) from various plant species and to an important group (group 13) of grass pollen allergens. The LLP-PG transcript is pollen-specific and it accumulates only at the latest stage during pollen development, in the mature pollen. In contrast to other "late genes" LLP-PG transcript can neither be induced by abscisic acid (ABA) nor by dehydration. Immunoblot analyses of pollen protein extracts from lily, timothy grass and tobacco with IgG antibodies directed against LLP-PG and against the timothy grass pollen allergen, Phl p 13, indicated that lily LLP-PG shares surface-exposed epitopes with pollen PGs from monocotyledonous and dicotyledonous plants. Enzyme-linked immunosorbent assay (ELISA) analyses and inhibition ELISA assays with patients' IgE demonstrated a very low IgE reactivity of lily rLLP-PG and a lack of cross-reactivity between rLLP-PG and the timothy grass pollen allergen, rPhl p 13. These data demonstrated that despite the significant sequence homology and the conserved surface-exposed epitopes LLP-PG represents a low-allergenic member of pollen PGs.

Allergens↗
PubMed2002

New pollen-specific receptor kinases identified in tomato, maize and Arabidopsis: the tomato kinases show overlapping but distinct localization patterns on pollen tubes.

We previously characterized LePRK1 and LePRK2, pollen-specific receptor kinases from tomato (Muschietti et al., 1998). Here we identify a similar receptor kinase from maize, ZmPRK1, that is also specifically expressed late in pollen development, and a third pollen receptor kinase from tomato, LePRK3. LePRK3 is less similar to LePRK1 and LePRK2 than either is to each other. We used immunolocalization to show that all three LePRKs localize to the pollen tube wall, in partially overlapping but distinct patterns. We used RT-PCR and degenerate primers to clone homologues of the tomato kinases from other Solanaceae. We deduced features diagnostic of pollen receptor kinases and used these criteria to identify family members in the Arabidopsis database. RT-PCR confirmed pollen expression for five of these Arabidopsis candidates; two of these are clearly homologues of LePRK3. Our results reveal the existence of a distinct pollen-specific receptor kinase gene family whose members are likely to be involved in perceiving extracellular cues during pollen tube growth.

Amino Acid Sequence↗
PubMed1972

In vitro measurement of pollen tube growth inhibition.

A method for estimating inhibition of pollen tube growth was developed. Pollen is placed in straight lines on an agar surface where it responds uniformly and predictably to aqueous solutions of germination-inhibiting substances located in wells at the ends of the lines. A scale of ratings, roughly corresponding to serial, doubled concentrations of inhibiting substances, was devised. Water-soluble organic solvents are relatively noninhibitory, salts are variable, and metabolic inhibitors have strong inhibitory effects. Pollens differ in their susceptibility to inhibition and in their response to particular substances.

Journal Article↗
PubMed1973

Development of the Generative Cell Wall in Monotropa uniflora L. Pollen.

During an ultrastructural survey of an achlorophyllus dicotyledonous plant, Monotropa uniflora L., a stage of pollen development was encountered which suggests a relationship between the activity of the rough endoplasmic reticulum and the development of the generative cell wall.

Journal Article↗
PubMed1985

Studies on Conditions for Cell Division and Embryogenesis in Isolated Pollen Culture of Nicotiana rustica.

A method for the induction of a high rate of cell division and embryogenesis of Nicotiana rustica pollen was developed. Binucleate pollen grains were fractionated by Percoll density gradient (35/45%) centrifugation and cultured in 0.4 molar mannitol at 30 degrees C (the first culture). After 3 days in culture pollen was recollected by a second Percoll fractionation (0/30%) and transferred to and cultured in a medium containing the Murashige-Skoog macro-elements, 0.4 molar mannitol, 40 millimolar galactose, 3 millimolar glutamine, and 5 micromolar ABA for 10 days (the second culture). The cell population consisting of about 80% dividing pollen was transferred to a Murashige-Skoog medium containing 0.4 molar mannitol, 3 millimolar glutamine, and no phytohormone (the third culture), where about 40% of dividing pollen developed into embryos or embryogenic calli.

Journal Article↗
PubMed2003

SPL8, an SBP-box gene that affects pollen sac development in Arabidopsis.

SQUAMOSA PROMOTER BINDING PROTEIN-box genes (SBP-box genes) encode plant-specific proteins that share a highly conserved DNA binding domain, the SBP domain. Although likely to represent transcription factors, little is known about their role in development. In Arabidopsis, SBP-box genes constitute a structurally heterogeneous family of 16 members known as SPL genes. For one of these genes, SPL8, we isolated three independent transposon-tagged mutants, all of which exhibited a strong reduction in fertility. Microscopic analysis revealed that this reduced fertility is attributable primarily to abnormally developed microsporangia, which exhibit premeiotic abortion of the sporocytes. In addition to its role in microsporogenesis, the SPL8 knockout also seems to affect megasporogenesis, trichome formation on sepals, and stamen filament elongation. The SPL8 mutants described help to uncover the roles of SBP-box genes in plant development.

Alleles↗
PubMedDate not supplied

[Development of pollen tubes in self-fertile and self-sterile lines of sugar beets in isolation].

Studies in pollen germination on stigmas of isolated plants showed a great difference in this process in different forms. A rapid and steady growth of pollen tubes, their accumulation near the micropyle and penetration in it are observed in self-fertile plants. Pollen tubes grow slowly, often forming thickenings and swellings in self-sterile forms. Some of them penetrate deeper, reaching sometimes the seedbud but turn back without penetrating into it.

Plants, Edible↗
PubMed2003

Proteome analysis of male gametophyte development in rice anthers.

We used proteomic analysis to investigate the changing patterns of protein synthesis during pollen development in anthers from rice plants grown under strictly controlled growth conditions. Cytological analysis and external growth measurements such as anther length, auricle distances and days before flowering were used to determine pollen developmental stages. This allowed the collection of synchronous anther materials representing six discrete pollen developmental stages. Proteins were extracted from the anther samples and separated by two-dimensional gel electrophoresis to produce proteome maps. The anther proteome maps of different developmental stages were compared and 150 protein spots, which were changed consistently during development, were analysed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry to produce peptide mass fingerprint (PMF) data. Database searches using these PMF data revealed the identities of 40 of the protein spots analyzed. These 40 proteins represent 33 unique gene products. Four protein spots that could not be identified by PMF analysis were analysed by N-terminal microsequencing. Multiple charge-isoforms of vacuolar acid invertase, fructokinase, beta-expansin and profilin were identified. These proteins are closely associated with sugar metabolism, cell elongation and cell expansion, all of which are cell activities that are essential to pollen germination. The existence of multiple isoforms of the same proteins suggests that during the process of pollen development some kind of post-translational modification of these proteins occurs.

Amino Acid Sequence↗
PubMed2002

Deletion of the C-terminal 138 amino acids of the wheat FKBP73 abrogates calmodulin binding, dimerization and male fertility in transgenic rice.

Wheat FKBP73 (wFKBP73) belongs to the FK506-binding protein (FKBP) family which, in common with the cyclophilin and parvulin families, possesses peptidyl prolyl cis-trans isomerase (PPIase) activity. Wheat FKBP73 has been shown to contain three FKBP12-like domains, a tetratricopeptide repeat (TPR) via which it binds heat shock protein 90 and a calmodulin-binding domain (CaMbd). In this study we investigated: (1) the contribution of the N-terminal and C-terminal moieties of wFKBP73 to its biological activity by over-expression of the prolyl isomerase domains in transgenic rice, and (2) the biochemical characteristics of the C-terminal moiety. The recombinant wFKBP73 was found to bind calmodulin via the CaMbd and to be present mainly as a dimer in solution. The dimerization was abrogated when 138 amino acids from the C-terminal half were deleted. Expression of the full-length FKBP73 produced fertile rice plants, whereas the expression of the peptidyl prolyl cis-trans isomerase domains in transgenic rice resulted in male-sterile plants. The male sterility was expressed at various stages of anther development with arrest of normal pollen development occurring after separation of the microspores from the tetrads. Although the direct cause of the dominant male sterility is not yet defined, we suggest that it is associated with a novel interaction of the prolyl isomerase domains with anther specific target proteins.

Binding Sites↗
PubMed1993

Characterization of a multigene family encoding an exopolygalacturonase in maize.

Genes coding for exopolygalacturonase in plants are abundantly expressed during the development of the male gametophyte (pollen). We have analysed genomic and cDNA clones for several representatives of the small multigene family encoding exopolygalacturonase from Zea mays. Structures for both actively transcribed genes and non-transcribed pseudogenes are reported. Comparisons of the nucleotide sequences for coding and flanking regions of different members of the gene family reveal surprisingly few base substitutions, suggesting that the exopolygalacturonase gene family of maize arose through very recent multiple duplication events. The pseudogenes are shown to possess an 80 bp insertion within the coding region, which may represent a relictual intron that has been lost in the active genes. We estimate that 12 exopolygalacturonase genes exist in maize. None appear to be expressed at a detectable level in tissue other than those associated with pollen development.

Base Sequence↗
PubMed2001

The PET1-CMS mitochondrial mutation in sunflower is associated with premature programmed cell death and cytochrome c release.

In mammals, mitochondria have been shown to play a key intermediary role in apoptosis, a morphologically distinct form of programmed cell death (PCD), for example, through the release of cytochrome c, which activates a proteolytic enzyme cascade, resulting in specific nuclear DNA degradation and cell death. In plants, PCD is a feature of normal development, including the penultimate stage of anther development, leading to dehiscence and pollen release. However, there is little evidence that plant mitochondria are involved in PCD. In a wide range of plant species, anther and/or pollen development is disrupted in a class of mutants termed CMS (for cytoplasmic male sterility), which is associated with mutations in the mitochondrial genome. On the basis of the manifestation of a number of morphological and biochemical markers of apoptosis, we have shown that the PET1-CMS cytoplasm in sunflower causes premature PCD of the tapetal cells, which then extends to other anther tissues. These features included cell condensation, oligonucleosomal cleavage of nuclear DNA, separation of chromatin into delineated masses, and initial persistence of mitochondria. In addition, immunocytochemical analysis revealed that cytochrome c was released partially from the mitochondria into the cytosol of tapetal cells before the gross morphological changes associated with PCD. The decrease in cytochrome c content in mitochondria isolated from male sterile florets preceded a decrease in the integrity of the outer mitochondrial membrane and respiratory control ratio. Our data suggest that plant mitochondria, like mammalian mitochondria, play a key role in the induction of PCD. The tissue-specific nature of the CMS phenotype is discussed with regard to cellular respiratory demand and PCD during normal anther development.

Cytochrome c Group↗