David TWELL | University of Leicester (original) (raw)
Papers by David TWELL
The Plant Cell, Apr 1, 2011
In plant cells, mitochondria and plastids contain their own genomes derived from the ancestral ba... more In plant cells, mitochondria and plastids contain their own genomes derived from the ancestral bacteria endosymbiont. Despite their limited genetic capacity, these multicopy organelle genomes account for a substantial fraction of total cellular DNA, raising the question of whether organelle DNA quantity is controlled spatially or temporally. In this study, we genetically dissected the organelle DNA decrease in pollen, a phenomenon that appears to be common in most angiosperm species. By staining mature pollen grains with fluorescent DNA dye, we screened Arabidopsis thaliana for mutants in which extrachromosomal DNAs had accumulated. Such a recessive mutant, termed defective in pollen organelle DNA degradation1 (dpd1), showing elevated levels of DNAs in both plastids and mitochondria, was isolated and characterized. DPD1 encodes a protein belonging to the exonuclease family, whose homologs appear to be found in angiosperms. Indeed, DPD1 has Mg2+-dependent exonuclease activity when expressed as a fusion protein and when assayed in vitro and is highly active in developing pollen. Consistent with the dpd phenotype, DPD1 is dual-targeted to plastids and mitochondria. Therefore, we provide evidence of active organelle DNA degradation in the angiosperm male gametophyte, primarily independent of maternal inheritance; the biological function of organellar DNA degradation in pollen is currently unclear.
Plant Physiology, May 1, 2002
Transcripts of the ntp303 gene accumulate abundantly throughout pollen development, whereas the p... more Transcripts of the ntp303 gene accumulate abundantly throughout pollen development, whereas the protein only accumulates to detectable levels after pollen germination. In an attempt to explain the divergence in the accumulation profiles of the mRNA and the protein, we investigated the role of the untranslated regions (UTRs) in enhancing ntp303 translation during the transition from developing to germinating pollen. Luciferase reporter gene fusion constructs containing the ntp303 5Ј-UTR gave rise to luciferase activity that was up to 60-fold higher during pollen tube growth than that of constructs containing different 5Ј-UTRs. No apparent differences in the luciferase activity of these constructs were observed during pollen development. The ntp303 5Ј-UTR-mediated increase in luciferase activity was not significantly influenced by coding region or 3Ј-UTR sequences. Furthermore, enhanced luciferase activity directed by the ntp303 5Ј-UTR occurred predominantly at the post-transcriptional level. A series of 5Ј-UTR deletion constructs was created to identify putative regulatory sequences required for the high level of translation during pollen tube growth. Two predicted stem loop structures (H-I and H-II) caused a complete inhibition of the enhanced translation after their total or partial deletion. A (GAA) 8 repeat within the H-I stem loop structure was demonstrated to be important for the modulation of translation efficiency. The H-II stem loop structure was found to be essential for the determination of mRNA stability.
Current Biology, Jun 1, 2012
<b>Copyright information:</b>Taken from "MADS-complexes regulate transcriptome d... more <b>Copyright information:</b>Taken from "MADS-complexes regulate transcriptome dynamics during pollen maturation"http://genomebiology.com/content/8/11/R249Genome Biology 2007;8(11):R249-R249.Published online 22 Nov 2007PMCID:PMC2258202. Of the genes downregulated in //triple mutant pollen, 83.4% exhibit peak expression at the mature pollen grain (MPG) stage of wild-type (WT) pollen development (according to Honys and Twell [11]), whereas 56.7% of the genes upregulated in this mutant peak during the immature tricellular stage (tricellular pollen [TCP]). In total, 83.5% of the upregulated genes peak during the three immature stages (unicellular microspores [UNM], BCP, and TCP). The AtMIKC* complexes contribute quite significantly to the transcriptional changes that occur during pollen maturation. We ranked all genes that were consistently called present in WT pollen according to their expression level in mature WT pollen, in descending order (the highest expressed gene received number 1). In this graph the ranking numbers of all genes upregulated and downregulated in triple mutant pollen are plotted, revealing that AtMIKC* complexes predominantly activate high-abundance and medium-abundance transcripts, while repressing low-abundance and medium-abundance transcripts. All calculations related to these graphs are included in Additional data file 2.
Plant Physiology Communications, 2010
We successfully cloned the full cDNA sequence of OsDUO1(Oryza sativa duo pollen 1) encoding a put... more We successfully cloned the full cDNA sequence of OsDUO1(Oryza sativa duo pollen 1) encoding a putative R2R3 type MYB transcription factor by RT-PCR.The OsDUO1 cDNA is 1 032 bp in length and encodes a protein with 343 amino acids.The expression of OsDUO1 was detectable only during the late pollen development in rice,suggesting the possible biological function of OsDUO1 during late rice pollen development.Bioinformatics analysis showed that OsDUO1 has close homologs in Brachypodium distachyon,Sorghum bicolor,Zea mays,Arabidopsis thaliana,Nicotiana tabacum,Vitis vinifera,Ricinus communis,Populus trichocarpa and Physcomitrella patens suggesting the conserved function of OsDUO1 in plants during evolution.
Biochemical Society Transactions, Mar 22, 2010
Pollen grains represent the highly reduced haploid male gametophyte generation in angiosperms. Th... more Pollen grains represent the highly reduced haploid male gametophyte generation in angiosperms. They play an essential role in plant fertility by generating and delivering twin sperm cells to the embryo sac to undergo double fertilization. The functional specialization of the male gametophyte and double fertilization are considered to be key innovations in the evolutionary success of angiosperms. The haploid nature of the male gametophyte and its highly tractable ontogeny makes it an attractive system to study many fundamental biological processes, such as cell fate determination, cell-cycle progression and gene regulation. The present mini-review encompasses key advances in our understanding of the molecular mechanisms controlling male gametophyte patterning in angiosperms. A brief overview of male gametophyte development is presented, followed by a discussion of the genes required at landmark events of male gametogenesis. The value of the male gametophyte as an experimental system to study the interplay between cell fate determination and cell-cycle progression is also discussed and exemplified with an emerging model outlining the regulatory networks that distinguish the fate of the male germline from its sister vegetative cell. We conclude with a perspective of the impact emerging data will have on future research strategies and how they will develop further our understanding of male gametogenesis and plant development.
Plant reproduction, Mar 12, 2015
Key message Overview of pollen transcriptome studies. Pollen development is driven by gene expres... more Key message Overview of pollen transcriptome studies. Pollen development is driven by gene expression, and knowledge of the molecular events underlying this process has undergone a quantum leap in the last decade through studies of the transcriptome. Here, we outline historical evidence for male haploid gene expression and review the wealth of pollen transcriptome data now available. Knowledge of the transcriptional capacity of pollen has progressed from genetic studies to the direct analysis of RNA and from gene-by-gene studies to analyses on a genomic scale. Microarray and/or RNA-seq data can now be accessed for all phases and cell types of developing pollen encompassing 10 different angiosperms. These growing resources have accelerated research and will undoubtedly inspire new directions and the application of system-based research into the mechanisms that govern the development, function and evolution of angiosperm pollen.
Journal of Plant Biology, May 25, 2022
Blackwell Publishing eBooks, 2002
ABSTRACT
Plant Signaling & Behavior, May 1, 2010
The tobacco MAP215/Dis1-family protein TMBP200 is required for the functional organization of mic... more The tobacco MAP215/Dis1-family protein TMBP200 is required for the functional organization of microtubule arrays during male germline establishment.
Proceedings of the National Academy of Sciences of the United States of America, Dec 8, 2014
Plant Journal, Jul 24, 2012
SummaryThe conserved Fused kinase plays vital but divergent roles in many organisms from Hedgehog... more SummaryThe conserved Fused kinase plays vital but divergent roles in many organisms from Hedgehog signalling in Drosophila to polarization and chemotaxis in Dictyostelium. Previously we have shown that Arabidopsis Fused kinase termed TWO‐IN‐ONE (TIO) is essential for cytokinesis in both sporophytic and gametophytic cell types. Here using in vivo imaging of GFP‐tagged microtubules in dividing microspores we show that TIO is required for expansion of the phragmoplast. We identify the phragmoplast‐associated kinesins, PAKRP1/Kinesin‐12A and PAKRP1L/Kinesin‐12B, as TIO‐interacting proteins and determine TIO‐Kinesin‐12 interaction domains and their requirement in male gametophytic cytokinesis. Our results support the role of TIO as a functional protein kinase that interacts with Kinesin‐12 subfamily members mainly through the C‐terminal ARM repeat domain, but with a contribution from the N‐terminal kinase domain. The interaction of TIO with Kinesin proteins and the functional requirement of their interaction domains support the operation of a Fused kinase signalling module in phragmoplast expansion that depends upon conserved structural features in diverse Fused kinases.
Journal of Experimental Botany, 2003
Plant Rac-like GTPases have been classi®ed phylogenetically into two major groupsÐclass I and cla... more Plant Rac-like GTPases have been classi®ed phylogenetically into two major groupsÐclass I and class II. Several pollen-expressed class I Rac-like GTPases have been shown to be important regulators of polar pollen tube growth. The functional participation by some of the class I and all of the class II Arabidopsis Rac-like GTPases in pollen tube growth remains to be explored. It is shown that at least four members of the Arabidopsis Rac GTPase family are expressed in pollen, including a class II Rac, AtRac7. However, when over-expressed as fusion proteins with GFP, both pollen-and non-pollen-expressed AtRacs interfered with the normal pollen tube tip growth process. These observations suggest that these AtRacs share similar biochemical activities and may integrate into the pollen cellular machinery that regulates the polar tube growth process. Therefore, the functional contribution by individual Rac GTPase to the pollen tube growth process probably depends to a considerable extent on their expression characteristics in pollen. Among the Arabidopsis Racs, GFP-AtRac7 showed association with the cell membrane and Golgi bodies, a pattern distinct from all previously reported localization for other plant Racs. Over-expressing GFP-AtRac7 also induced the broadest spectrum of pollen tube growth defects, including pollen tubes that are bifurcated, with diverted growth trajectory or a ballooned tip. Transgenic plants with multiple copies of the chimeric Lat52-GFP-AtRac7 showed severely reduced seed set, probably many of these defective pollen tubes were arrested, or reduced in their growth rates that they did not arrive at the ovules while they were still receptive for fertilization. These observations substantiate the importance of Rac-like GTPases to sexual reproduction.
![Research paper thumbnail of <i>SETH1</i>and<i>SETH2</i>, Two Components of the Glycosylphosphatidylinositol Anchor Biosynthetic Pathway, Are Required for Pollen Germination and Tube Growth in Arabidopsis [W]](https://attachments.academia-assets.com/112256727/thumbnails/1.jpg)
](The Plant Cell, 2004
Glycosylphosphatidylinositol (GPI) anchoring provides an alternative to transmembrane domains for... more Glycosylphosphatidylinositol (GPI) anchoring provides an alternative to transmembrane domains for anchoring proteins to the cell surface in eukaryotes. GPI anchors are synthesized in the endoplasmic reticulum via the sequential addition of monosaccharides, fatty acids, and phosphoethanolamines to phosphatidylinositol. Deficiencies in GPI biosynthesis lead to embryonic lethality in animals and to conditional lethality in eukaryotic microbes by blocking cell growth, cell division, or morphogenesis. We report the genetic and phenotypic analysis of insertional mutations disrupting SETH1 and SETH2, which encode Arabidopsis homologs of two conserved proteins involved in the first step of the GPI biosynthetic pathway. seth1 and seth2 mutations specifically block male transmission and pollen function. This results from reduced pollen germination and tube growth, which are associated with abnormal callose deposition. This finding suggests an essential role for GPI anchor biosynthesis in pollen tube wall deposition or metabolism. Using transcriptomic and proteomic approaches, we identified 47 genes that encode potential GPI-anchored proteins that are expressed in pollen and demonstrated that at least 11 of these proteins are associated with pollen membranes by GPI anchoring. Many of the identified candidate proteins are homologous with proteins involved in cell wall synthesis and remodeling or intercellular signaling and adhesion, and they likely play important roles in the establishment and maintenance of polarized pollen tube growth.
Development, Jul 1, 1990
To investigate the regulation of gene expression during male gametophyte development, we analyzed... more To investigate the regulation of gene expression during male gametophyte development, we analyzed the promoter activity of two different genes (LAT52 and LAT59) from tomato, isolated on the basis of their anther-specific expression. In transgenic tomato, tobacco and Arabidopsis plants containing the LAT52 promoter region fused to the /3-glucuronidase (GUS) gene, GUS activity was restricted to pollen. Transgenic tomato, tobacco and Arabidopsis plants containing the LAT59 promoter region fused to GUS also showed very high levels of GUS activity in pollen. However, low levels of expression of the LAT59 promoter construct were also detected in seeds and roots. With both constructs, the appearance of GUS activity in developing anthers was correlated with the onset of microspore mitosis and increased progressively until anthesis (pollen shed). Our results demonstrate coordinate regulation of the LAT52 and LAT59 promoters in developing microspores and suggest that the mechanisms that regulate pollen-specific gene expression are evolutionarily conserved.
Annual Plant Reviews online, Apr 19, 2018
Springer eBooks, 1991
The development of pollen as a vector for direct gene transfer would be a significant advance in ... more The development of pollen as a vector for direct gene transfer would be a significant advance in our ability to introduce genes into plants. Such methodology should be of general utility for many plant species, and in particular for the major monocotyledonous crop plants such as maize, wheat and barley that are recalcitrant to protoplast regeneration and that are not amenable to Agrobacterium based transformation techniques. A further advantage would be the avoidance of tissue culture steps that are time consuming and known to result in undesirable somaclonal variation. The potential of pollen as a vector for direct gene transfer has long been realized. For more than 10 years numerous investigators have attempted pollen-mediated transformation, several of which have claimed success [5, 8, 19, 23]. However the ultimate proof that transformation has taken place, that is, the demonstration of integration of foreign DNA into the nuclear genome at the molecular level and the genetic transmission of this DNA, is still lacking. This chapter presents a summary of research that has been directed towards pollen-mediated gene transfer, a detailed protocol for the delivery of DNA into pollen using particle bombardment and a discussion of factors that may be important for the successful application of this technique to obtain stably transformed plants.
In prokaryotic and eukaryotic organisms asymmetry in the fate of daughter cells is commonly estab... more In prokaryotic and eukaryotic organisms asymmetry in the fate of daughter cells is commonly established by the unequal division of intrinsically polar mother cells. First microspore division represents a striking example of such an intrisically asymmetric division which has dramatic and determinative consequences for the differentiation or fate of the two unequal daughter cells. The aims of this chapter are to discuss the significance of asymmetric cell division for correct pollen differentiation, how asymmetric division leads to differential cell fate, how microspores develop the necessary polarity required for asymmetric division, and to summarize the results of new approaches that are being adopted to identify key genes involved in these processes.
Plant Journal, Jul 1, 2016
In flowering plants, male gametes arise via meiosis of diploid pollen mother cells followed by tw... more In flowering plants, male gametes arise via meiosis of diploid pollen mother cells followed by two rounds of mitotic division. Haploid microspores undergo polar nuclear migration and asymmetric division at pollen mitosis I to segregate the male germline, followed by division of the germ cell to generate a pair of sperm cells. We previously reported two gemini pollen (gem) mutants that produced twin-celled pollen arising from polarity and cytokinesis defects at pollen mitosis I in Arabidopsis. Here, we report an independent mutant, gem3, with a similar division phenotype and severe genetic transmission defects through pollen. Cytological analyses revealed that gem3 disrupts cell division during male meiosis, at pollen mitosis I and during female gametophyte development. We show that gem3 is a hypomorphic allele (aug6-1) of AUGMIN subunit 6, encoding a conserved component in the augmin complex, which mediates microtubule (MT)dependent MT nucleation in acentrosomal cells. We show that MT arrays are disturbed in gem3/aug6-1 during male meiosis and pollen mitosis I using fluorescent MT-markers. Our results demonstrate a broad role for the augmin complex in MT organization during sexual reproduction, and highlight gem3/aug6-1 mutants as a valuable tool for the investigation of augmin-dependent MT nucleation and dynamics in plant cells.
The Plant Cell, Apr 1, 2011
In plant cells, mitochondria and plastids contain their own genomes derived from the ancestral ba... more In plant cells, mitochondria and plastids contain their own genomes derived from the ancestral bacteria endosymbiont. Despite their limited genetic capacity, these multicopy organelle genomes account for a substantial fraction of total cellular DNA, raising the question of whether organelle DNA quantity is controlled spatially or temporally. In this study, we genetically dissected the organelle DNA decrease in pollen, a phenomenon that appears to be common in most angiosperm species. By staining mature pollen grains with fluorescent DNA dye, we screened Arabidopsis thaliana for mutants in which extrachromosomal DNAs had accumulated. Such a recessive mutant, termed defective in pollen organelle DNA degradation1 (dpd1), showing elevated levels of DNAs in both plastids and mitochondria, was isolated and characterized. DPD1 encodes a protein belonging to the exonuclease family, whose homologs appear to be found in angiosperms. Indeed, DPD1 has Mg2+-dependent exonuclease activity when expressed as a fusion protein and when assayed in vitro and is highly active in developing pollen. Consistent with the dpd phenotype, DPD1 is dual-targeted to plastids and mitochondria. Therefore, we provide evidence of active organelle DNA degradation in the angiosperm male gametophyte, primarily independent of maternal inheritance; the biological function of organellar DNA degradation in pollen is currently unclear.
Plant Physiology, May 1, 2002
Transcripts of the ntp303 gene accumulate abundantly throughout pollen development, whereas the p... more Transcripts of the ntp303 gene accumulate abundantly throughout pollen development, whereas the protein only accumulates to detectable levels after pollen germination. In an attempt to explain the divergence in the accumulation profiles of the mRNA and the protein, we investigated the role of the untranslated regions (UTRs) in enhancing ntp303 translation during the transition from developing to germinating pollen. Luciferase reporter gene fusion constructs containing the ntp303 5Ј-UTR gave rise to luciferase activity that was up to 60-fold higher during pollen tube growth than that of constructs containing different 5Ј-UTRs. No apparent differences in the luciferase activity of these constructs were observed during pollen development. The ntp303 5Ј-UTR-mediated increase in luciferase activity was not significantly influenced by coding region or 3Ј-UTR sequences. Furthermore, enhanced luciferase activity directed by the ntp303 5Ј-UTR occurred predominantly at the post-transcriptional level. A series of 5Ј-UTR deletion constructs was created to identify putative regulatory sequences required for the high level of translation during pollen tube growth. Two predicted stem loop structures (H-I and H-II) caused a complete inhibition of the enhanced translation after their total or partial deletion. A (GAA) 8 repeat within the H-I stem loop structure was demonstrated to be important for the modulation of translation efficiency. The H-II stem loop structure was found to be essential for the determination of mRNA stability.
Current Biology, Jun 1, 2012
<b>Copyright information:</b>Taken from "MADS-complexes regulate transcriptome d... more <b>Copyright information:</b>Taken from "MADS-complexes regulate transcriptome dynamics during pollen maturation"http://genomebiology.com/content/8/11/R249Genome Biology 2007;8(11):R249-R249.Published online 22 Nov 2007PMCID:PMC2258202. Of the genes downregulated in //triple mutant pollen, 83.4% exhibit peak expression at the mature pollen grain (MPG) stage of wild-type (WT) pollen development (according to Honys and Twell [11]), whereas 56.7% of the genes upregulated in this mutant peak during the immature tricellular stage (tricellular pollen [TCP]). In total, 83.5% of the upregulated genes peak during the three immature stages (unicellular microspores [UNM], BCP, and TCP). The AtMIKC* complexes contribute quite significantly to the transcriptional changes that occur during pollen maturation. We ranked all genes that were consistently called present in WT pollen according to their expression level in mature WT pollen, in descending order (the highest expressed gene received number 1). In this graph the ranking numbers of all genes upregulated and downregulated in triple mutant pollen are plotted, revealing that AtMIKC* complexes predominantly activate high-abundance and medium-abundance transcripts, while repressing low-abundance and medium-abundance transcripts. All calculations related to these graphs are included in Additional data file 2.
Plant Physiology Communications, 2010
We successfully cloned the full cDNA sequence of OsDUO1(Oryza sativa duo pollen 1) encoding a put... more We successfully cloned the full cDNA sequence of OsDUO1(Oryza sativa duo pollen 1) encoding a putative R2R3 type MYB transcription factor by RT-PCR.The OsDUO1 cDNA is 1 032 bp in length and encodes a protein with 343 amino acids.The expression of OsDUO1 was detectable only during the late pollen development in rice,suggesting the possible biological function of OsDUO1 during late rice pollen development.Bioinformatics analysis showed that OsDUO1 has close homologs in Brachypodium distachyon,Sorghum bicolor,Zea mays,Arabidopsis thaliana,Nicotiana tabacum,Vitis vinifera,Ricinus communis,Populus trichocarpa and Physcomitrella patens suggesting the conserved function of OsDUO1 in plants during evolution.
Biochemical Society Transactions, Mar 22, 2010
Pollen grains represent the highly reduced haploid male gametophyte generation in angiosperms. Th... more Pollen grains represent the highly reduced haploid male gametophyte generation in angiosperms. They play an essential role in plant fertility by generating and delivering twin sperm cells to the embryo sac to undergo double fertilization. The functional specialization of the male gametophyte and double fertilization are considered to be key innovations in the evolutionary success of angiosperms. The haploid nature of the male gametophyte and its highly tractable ontogeny makes it an attractive system to study many fundamental biological processes, such as cell fate determination, cell-cycle progression and gene regulation. The present mini-review encompasses key advances in our understanding of the molecular mechanisms controlling male gametophyte patterning in angiosperms. A brief overview of male gametophyte development is presented, followed by a discussion of the genes required at landmark events of male gametogenesis. The value of the male gametophyte as an experimental system to study the interplay between cell fate determination and cell-cycle progression is also discussed and exemplified with an emerging model outlining the regulatory networks that distinguish the fate of the male germline from its sister vegetative cell. We conclude with a perspective of the impact emerging data will have on future research strategies and how they will develop further our understanding of male gametogenesis and plant development.
Plant reproduction, Mar 12, 2015
Key message Overview of pollen transcriptome studies. Pollen development is driven by gene expres... more Key message Overview of pollen transcriptome studies. Pollen development is driven by gene expression, and knowledge of the molecular events underlying this process has undergone a quantum leap in the last decade through studies of the transcriptome. Here, we outline historical evidence for male haploid gene expression and review the wealth of pollen transcriptome data now available. Knowledge of the transcriptional capacity of pollen has progressed from genetic studies to the direct analysis of RNA and from gene-by-gene studies to analyses on a genomic scale. Microarray and/or RNA-seq data can now be accessed for all phases and cell types of developing pollen encompassing 10 different angiosperms. These growing resources have accelerated research and will undoubtedly inspire new directions and the application of system-based research into the mechanisms that govern the development, function and evolution of angiosperm pollen.
Journal of Plant Biology, May 25, 2022
Blackwell Publishing eBooks, 2002
ABSTRACT
Plant Signaling & Behavior, May 1, 2010
The tobacco MAP215/Dis1-family protein TMBP200 is required for the functional organization of mic... more The tobacco MAP215/Dis1-family protein TMBP200 is required for the functional organization of microtubule arrays during male germline establishment.
Proceedings of the National Academy of Sciences of the United States of America, Dec 8, 2014
Plant Journal, Jul 24, 2012
SummaryThe conserved Fused kinase plays vital but divergent roles in many organisms from Hedgehog... more SummaryThe conserved Fused kinase plays vital but divergent roles in many organisms from Hedgehog signalling in Drosophila to polarization and chemotaxis in Dictyostelium. Previously we have shown that Arabidopsis Fused kinase termed TWO‐IN‐ONE (TIO) is essential for cytokinesis in both sporophytic and gametophytic cell types. Here using in vivo imaging of GFP‐tagged microtubules in dividing microspores we show that TIO is required for expansion of the phragmoplast. We identify the phragmoplast‐associated kinesins, PAKRP1/Kinesin‐12A and PAKRP1L/Kinesin‐12B, as TIO‐interacting proteins and determine TIO‐Kinesin‐12 interaction domains and their requirement in male gametophytic cytokinesis. Our results support the role of TIO as a functional protein kinase that interacts with Kinesin‐12 subfamily members mainly through the C‐terminal ARM repeat domain, but with a contribution from the N‐terminal kinase domain. The interaction of TIO with Kinesin proteins and the functional requirement of their interaction domains support the operation of a Fused kinase signalling module in phragmoplast expansion that depends upon conserved structural features in diverse Fused kinases.
Journal of Experimental Botany, 2003
Plant Rac-like GTPases have been classi®ed phylogenetically into two major groupsÐclass I and cla... more Plant Rac-like GTPases have been classi®ed phylogenetically into two major groupsÐclass I and class II. Several pollen-expressed class I Rac-like GTPases have been shown to be important regulators of polar pollen tube growth. The functional participation by some of the class I and all of the class II Arabidopsis Rac-like GTPases in pollen tube growth remains to be explored. It is shown that at least four members of the Arabidopsis Rac GTPase family are expressed in pollen, including a class II Rac, AtRac7. However, when over-expressed as fusion proteins with GFP, both pollen-and non-pollen-expressed AtRacs interfered with the normal pollen tube tip growth process. These observations suggest that these AtRacs share similar biochemical activities and may integrate into the pollen cellular machinery that regulates the polar tube growth process. Therefore, the functional contribution by individual Rac GTPase to the pollen tube growth process probably depends to a considerable extent on their expression characteristics in pollen. Among the Arabidopsis Racs, GFP-AtRac7 showed association with the cell membrane and Golgi bodies, a pattern distinct from all previously reported localization for other plant Racs. Over-expressing GFP-AtRac7 also induced the broadest spectrum of pollen tube growth defects, including pollen tubes that are bifurcated, with diverted growth trajectory or a ballooned tip. Transgenic plants with multiple copies of the chimeric Lat52-GFP-AtRac7 showed severely reduced seed set, probably many of these defective pollen tubes were arrested, or reduced in their growth rates that they did not arrive at the ovules while they were still receptive for fertilization. These observations substantiate the importance of Rac-like GTPases to sexual reproduction.
The Plant Cell, 2004
Glycosylphosphatidylinositol (GPI) anchoring provides an alternative to transmembrane domains for... more Glycosylphosphatidylinositol (GPI) anchoring provides an alternative to transmembrane domains for anchoring proteins to the cell surface in eukaryotes. GPI anchors are synthesized in the endoplasmic reticulum via the sequential addition of monosaccharides, fatty acids, and phosphoethanolamines to phosphatidylinositol. Deficiencies in GPI biosynthesis lead to embryonic lethality in animals and to conditional lethality in eukaryotic microbes by blocking cell growth, cell division, or morphogenesis. We report the genetic and phenotypic analysis of insertional mutations disrupting SETH1 and SETH2, which encode Arabidopsis homologs of two conserved proteins involved in the first step of the GPI biosynthetic pathway. seth1 and seth2 mutations specifically block male transmission and pollen function. This results from reduced pollen germination and tube growth, which are associated with abnormal callose deposition. This finding suggests an essential role for GPI anchor biosynthesis in pollen tube wall deposition or metabolism. Using transcriptomic and proteomic approaches, we identified 47 genes that encode potential GPI-anchored proteins that are expressed in pollen and demonstrated that at least 11 of these proteins are associated with pollen membranes by GPI anchoring. Many of the identified candidate proteins are homologous with proteins involved in cell wall synthesis and remodeling or intercellular signaling and adhesion, and they likely play important roles in the establishment and maintenance of polarized pollen tube growth.
Development, Jul 1, 1990
To investigate the regulation of gene expression during male gametophyte development, we analyzed... more To investigate the regulation of gene expression during male gametophyte development, we analyzed the promoter activity of two different genes (LAT52 and LAT59) from tomato, isolated on the basis of their anther-specific expression. In transgenic tomato, tobacco and Arabidopsis plants containing the LAT52 promoter region fused to the /3-glucuronidase (GUS) gene, GUS activity was restricted to pollen. Transgenic tomato, tobacco and Arabidopsis plants containing the LAT59 promoter region fused to GUS also showed very high levels of GUS activity in pollen. However, low levels of expression of the LAT59 promoter construct were also detected in seeds and roots. With both constructs, the appearance of GUS activity in developing anthers was correlated with the onset of microspore mitosis and increased progressively until anthesis (pollen shed). Our results demonstrate coordinate regulation of the LAT52 and LAT59 promoters in developing microspores and suggest that the mechanisms that regulate pollen-specific gene expression are evolutionarily conserved.
Annual Plant Reviews online, Apr 19, 2018
Springer eBooks, 1991
The development of pollen as a vector for direct gene transfer would be a significant advance in ... more The development of pollen as a vector for direct gene transfer would be a significant advance in our ability to introduce genes into plants. Such methodology should be of general utility for many plant species, and in particular for the major monocotyledonous crop plants such as maize, wheat and barley that are recalcitrant to protoplast regeneration and that are not amenable to Agrobacterium based transformation techniques. A further advantage would be the avoidance of tissue culture steps that are time consuming and known to result in undesirable somaclonal variation. The potential of pollen as a vector for direct gene transfer has long been realized. For more than 10 years numerous investigators have attempted pollen-mediated transformation, several of which have claimed success [5, 8, 19, 23]. However the ultimate proof that transformation has taken place, that is, the demonstration of integration of foreign DNA into the nuclear genome at the molecular level and the genetic transmission of this DNA, is still lacking. This chapter presents a summary of research that has been directed towards pollen-mediated gene transfer, a detailed protocol for the delivery of DNA into pollen using particle bombardment and a discussion of factors that may be important for the successful application of this technique to obtain stably transformed plants.
In prokaryotic and eukaryotic organisms asymmetry in the fate of daughter cells is commonly estab... more In prokaryotic and eukaryotic organisms asymmetry in the fate of daughter cells is commonly established by the unequal division of intrinsically polar mother cells. First microspore division represents a striking example of such an intrisically asymmetric division which has dramatic and determinative consequences for the differentiation or fate of the two unequal daughter cells. The aims of this chapter are to discuss the significance of asymmetric cell division for correct pollen differentiation, how asymmetric division leads to differential cell fate, how microspores develop the necessary polarity required for asymmetric division, and to summarize the results of new approaches that are being adopted to identify key genes involved in these processes.
Plant Journal, Jul 1, 2016
In flowering plants, male gametes arise via meiosis of diploid pollen mother cells followed by tw... more In flowering plants, male gametes arise via meiosis of diploid pollen mother cells followed by two rounds of mitotic division. Haploid microspores undergo polar nuclear migration and asymmetric division at pollen mitosis I to segregate the male germline, followed by division of the germ cell to generate a pair of sperm cells. We previously reported two gemini pollen (gem) mutants that produced twin-celled pollen arising from polarity and cytokinesis defects at pollen mitosis I in Arabidopsis. Here, we report an independent mutant, gem3, with a similar division phenotype and severe genetic transmission defects through pollen. Cytological analyses revealed that gem3 disrupts cell division during male meiosis, at pollen mitosis I and during female gametophyte development. We show that gem3 is a hypomorphic allele (aug6-1) of AUGMIN subunit 6, encoding a conserved component in the augmin complex, which mediates microtubule (MT)dependent MT nucleation in acentrosomal cells. We show that MT arrays are disturbed in gem3/aug6-1 during male meiosis and pollen mitosis I using fluorescent MT-markers. Our results demonstrate a broad role for the augmin complex in MT organization during sexual reproduction, and highlight gem3/aug6-1 mutants as a valuable tool for the investigation of augmin-dependent MT nucleation and dynamics in plant cells.