Cell Biophysics and Development
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Research at the Plant Development group is concerned with developing integrated models of cellular growth and morphogenesis using the pollen tube as a biological model, ion dynamics as an experimental paradigm and theoretical modeling as an integrative tool. We have characterized novel ion channels involved in pollen tube growth and morphogenesis, by means of imaging, electrophysiology, genetics and molecular biology, and, furthermore, unravelled some of thegenetic and cellular mechanisms behind pollen tube guidance and fertilization in plants. Our group has provided novel insight into the transcriptional status of plant male gametes and its consequences for plant reproduction and improvement. The members of the group have been pivotal in the strengthening of the imaging, transcriptomics and vibrating probe electrophysiology services of the IGC. We have several collaboration with theoretical, animal developmental, evo-devo and other groups at the IGC, apart form external collaborations with research groups on Portugal and abroad.
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- Reprogramming of DNA Methylation in Pollen Guides Epigenetic Inheritance via Small RNAPublication . Calarco, Joseph P.; Borges, Filipe; Donoghue, Mark T.A.; Van Ex, Frédéric; Jullien, Pauline E.; Lopes, Telma; Gardner, Rui; Berger, Frédéric; Feijó, José A.; Becker, Jörg D.; Martienssen, Robert A.Epigenetic inheritance is more widespread in plants than in mammals, in part because mammals erase epigenetic information by germline reprogramming. We sequenced the methylome of three haploid cell types from developing pollen: the sperm cell, the vegetative cell, and their precursor, the postmeiotic microspore, and found that unlike in mammals the plant germline retains CG and CHG DNA methylation. However, CHH methylation is lost from retrotransposons in microspores and sperm cells and restored by de novo DNA methyltransferase guided by 24 nt small interfering RNA, both in the vegetative nucleus and in the embryo after fertilization. In the vegetative nucleus, CG methylation is lost from targets of DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), and their homologs, which include imprinted loci and recurrent epialleles that accumulate corresponding small RNA and are premethylated in sperm. Thus genome reprogramming in pollen contributes to epigenetic inheritance, transposon silencing, and imprinting, guided by small RNA.
- Transcriptional profiling of Arabidopsis root hairs and pollen defines an apical cell growth signaturePublication . Becker, Jörg D; Takeda, Seiji; Borges, Filipe; Dolan, Liam; Feijó, José ACurrent views on the control of cell development are anchored on the notion that phenotypes are defined by networks of transcriptional activity. The large amounts of information brought about by transcriptomics should allow the definition of these networks through the analysis of cell-specific transcriptional signatures. Here we test this principle by applying an analogue to comparative anatomy at the cellular level, searching for conserved transcriptional signatures, or conserved small gene-regulatory networks (GRNs) on root hairs (RH) and pollen tubes (PT), two filamentous apical growing cells that are a striking example of conservation of structure and function in plants.
- GABA signalling modulates plant growth by directly regulating the activity of plant-specific anion transportersPublication . Ramesh, Sunita A.; Tyerman, Stephen D.; Xu, Bo; Bose, Jayakumar; Kaur, Satwinder; Conn, Vanessa; Domingos, Patricia; Ullah, Sana; Wege, Stefanie; Shabala, Sergey; Feijó, José A.; Ryan, Peter R.; Gillham, MatthewThe non-protein amino acid, gamma-aminobutyric acid (GABA) rapidly accumulates in plant tissues in response to biotic and abiotic stress, and regulates plant growth. Until now it was not known whether GABA exerts its effects in plants through the regulation of carbon metabolism or via an unidentified signalling pathway. Here, we demonstrate that anion flux through plant aluminium-activated malate transporter (ALMT) proteins is activated by anions and negatively regulated by GABA. Site-directed mutagenesis of selected amino acids within ALMT proteins abolishes GABA efficacy but does not alter other transport properties. GABA modulation of ALMT activity results in altered root growth and altered root tolerance to alkaline pH, acid pH and aluminium ions. We propose that GABA exerts its multiple physiological effects in plants via ALMT, including the regulation of pollen tube and root growth, and that GABA can finally be considered a legitimate signalling molecule in both the plant and animal kingdoms.
- A Transcriptome Atlas of Physcomitrella patens Provides Insights into the Evolution and Development of Land PlantsPublication . Ortiz-Ramírez, Carlos; Hernandez-Coronado, Marcela; Thamm, Anna; Catarino, Bruno; Wang, Mingyi; Dolan, Liam; Feijó, José A.; Becker, Jörg D.Identifying the genetic mechanisms that underpin the evolution of new organ and tissue systems is an aim of evolutionary developmental biology. Comparative functional genetic studies between angiosperms and bryophytes can define those genetic changes that were responsible for developmental innovations. Here, we report the generation of a transcriptome atlas covering most phases in the life cycle of the model bryophyte Physcomitrella patens, including detailed sporophyte developmental progression. We identified a comprehensive set of sporophyte-specific transcription factors, and found that many of these genes have homologs in angiosperms that function in developmental processes such as flowering and shoot branching. Deletion of the PpTCP5 transcription factor results in development of supernumerary sporangia attached to a single seta, suggesting that it negatively regulates branching in the moss sporophyte. Given that TCP genes repress branching in angiosperms, we suggest that this activity is ancient. Finally, comparison of P. patens and Arabidopsis thaliana transcriptomes led us to the identification of a conserved core of transcription factors expressed in tip-growing cells. We identified modifications in the expression patterns of these genes that could account for developmental differences between P. patens tip-growing cells and A. thaliana pollen tubes and root hairs.
- Signaling with Ions: The Keystone for Apical Cell Growth and Morphogenesis in Pollen TubesPublication . Michard, Erwan; Simon, Alexander A.; Tavares, Bárbara; Wudick, Michael M.; Feijó, José A
- Molecular and electrophysiological characterization of anion transport in Arabidopsis thaliana pollen reveals regulatory roles for pH, Ca2+ and GABAPublication . Domingos, Patrícia; Dias, Pedro N; Tavares, Bárbara; Portes, Maria Teresa; Wudick, Michael M; Konrad, Kai R; Gilliham, Matthew; Bicho, Ana; Feijó, José AWe investigated the molecular basis and physiological implications of anion transport during pollen tube (PT) growth in Arabidopsis thaliana (Col-0). Patch-clamp whole-cell configuration analysis of pollen grain protoplasts revealed three subpopulations of anionic currents differentially regulated by cytoplasmic calcium ([Ca2+ ]cyt ). We investigated the pollen-expressed proteins AtSLAH3, AtALMT12, AtTMEM16 and AtCCC as the putative anion transporters responsible for these currents. AtCCC-GFP was observed at the shank and AtSLAH3-GFP at the tip and shank of the PT plasma membrane. Both are likely to carry the majority of anion current at negative potentials, as extracellular anionic fluxes measured at the tip of PTs with an anion vibrating probe were significantly lower in slah3-/- and ccc-/- mutants, but unaffected in almt12-/- and tmem16-/- . We further characterised the effect of pH and GABA by patch clamp. Strong regulation by extracellular pH was observed in the wild-type, but not in tmem16-/- . Our results are compatible with AtTMEM16 functioning as an anion/H+ cotransporter and therefore, as a putative pH sensor. GABA presence: (1) inhibited the overall currents, an effect that is abrogated in the almt12-/- and (2) reduced the current in AtALMT12 transfected COS-7 cells, strongly suggesting the direct interaction of GABA with AtALMT12. Our data show that AtSLAH3 and AtCCC activity is sufficient to explain the major component of extracellular anion fluxes, and unveils a possible regulatory system linking PT growth modulation by pH, GABA, and [Ca2+ ]cyt through anionic transporters.
- Plasma membrane H+-ATPases sustain pollen tube growth and fertilizationPublication . Hoffmann, Robert D.; Portes, Maria Teresa; Olsen, Lene Irene; Damineli, Daniel Santa Cruz; Hayashi, Maki; Nunes, Custódio O.; Pedersen, Jesper T.; Lima, Pedro T.; Campos, Cláudia; Feijó, José A.; Palmgren, MichaelPollen tubes are highly polarized tip-growing cells that depend on cytosolic pH gradients for signaling and growth. Autoinhibited plasma membrane proton (H+) ATPases (AHAs) have been proposed to energize pollen tube growth and underlie cell polarity, however, mechanistic evidence for this is lacking. Here we report that the combined loss of AHA6, AHA8, and AHA9 in Arabidopsis thaliana delays pollen germination and causes pollen tube growth defects, leading to drastically reduced fertility. Pollen tubes of aha mutants had reduced extracellular proton (H+) and anion fluxes, reduced cytosolic pH, reduced tip-to-shank proton gradients, and defects in actin organization. Furthermore, mutant pollen tubes had less negative membrane potentials, substantiating a mechanistic role for AHAs in pollen tube growth through plasma membrane hyperpolarization. Our findings define AHAs as energy transducers that sustain the ionic circuit defining the spatial and temporal profiles of cytosolic pH, thereby controlling downstream pH-dependent mechanisms essential for pollen tube elongation, and thus plant fertility.