Summary of Research Activities

(1) Reproductive Plant Biology - Signal Perception and Transduction

In the past 2 decades the research group has focused its main interest on polarity mechanisms and how extracellular signals are perceived and intracellularly transduced. Using pollen tubes as a biological model, we tried to dissect the signalling cascade involved in cell guidance; this is particularly relevant in angiosperm development since successful fertilisation depends on this accomplishment.

Having the animal paradigm as a background information, we proceed to determine (1) a series of stimuli to which the cells respond, (2) the intracellular Ca²⁺ response associated with these stimuli, (3) the positional elements - proteins - responsible for signal perception and (4) the target molecules involved in the transduction of the signal. These studies were performed with state-of-the-art imaging technology in living cells, namely Laser Scanning Confocal, photometry, wide-field video microscopy, caged-probes and microinjection of recombinant labelled proteins and mRNA.

Through application of external weak electrical fields and release of caged-Ca²⁺, we were the first to demonstrate that cytosolic free Ca²⁺ acts as a 2^nd messenger in pollen tube guidance (Plant J. 5: 331-341). Using a dye-quenching technique, we proceed to demonstrate that asymmetric Ca²⁺ channel activity (higher in the apical region) is responsible for the perception of extracellular signalling cues (Plant Cell 7: 1173-1184), an indication that pollen tube guidance is mediated by these proteins. We then showed that localised oscillations of cytosolic free Ca²⁺ in the apical region control growth directioning (Plant Cell 8: 1935-1949). This was one of the first studies where manipulation of intracellular Ca²⁺ levels in tip growing cells was achieved.

We imaged the distribution of calmodulin (both the protein and its mRNA) during pollen tube growth and reorientation (Sex Plant Reprod. 11: 131-139). In opposition to what was obtained with fixed cells, we observed that the protein distributed evenly in the cell. However, when the concentration of exogenous calmodulin was raised above the endogenous pool, a putative interaction with the actin cytoskeleton could be visualised. Although strict threshold conditions were used in this experiment, it nevertheless suggests the localisation of potential targets for calmodulin regulation in polarised growth.

Calmodulin activity seems however to be higher in the apex (Plant J. 38: 887-897) as demonstrated by ratio imaging of TA-CaM, a fluorescent analogue of the protein. The activity of the protein seems to be modulated by Ca²⁺ since it oscillates with a periodicity similar to Ca²⁺.

Using caged-probe technology we also obtained evidence in plant cells of an asymmetric functionality of Ins(1,4,5)P₃ receptors (Sex Plant Reprod. 11: 231-235) thus bringing closer animal and plant cells in which concerns the signal transduction pathways.

We imaged Ca²⁺-dependent protein kinase activity using a procedure which allowed measurements in live cells. It was found that protein kinase activity controls tube growth possibly by interaction with the Ca²⁺ channels (Plant Cell 10: 1499-1510).

We have also analysed the role of cAMP in pollen tube growth (Proc. Natl. Acad. Sci. USA 98: 10481-10486). Using FRET imaging of 3’,5’-cyclicAMP (cAMP) distribution in living pollen tubes microinjected with the PKA-derived fluorosensor, we recorded a uniform distribution of cAMP with a resting concentration of ~100-150 nM. Changes in cAMP arise through the activity of a putative adenylate cyclase identified in pollen. Antisense assays performed on growing pollen tubes with oligonucleotide probes (Sex. Plant Reprod., 14: 101-104) directed against conserved motifs transiently perturbed tip growth.

As a result of a collaboration with the group of J. Harper (Univ of Nevada) we published a significant contribution on  the role of cyclic nucleotide gated channels  (7 and 8) and their essential role for male fertility (PLOS one 8(2):e55277).

Another research project started by this group deals with the study of endo/exocytosis in tube growth and how this process is modulated by signal transduction pathways.  The data obtained indicates that endo/exocytosis is a main end-target of most signalling pathways already identified in pollen tubes: Ca²⁺ (Plant Sig. & Behavior, 1: 152-157), Rop GTPases (J. Exptl. Bot., 54: 83-92), cAMP and calmodulin (Plant J. 38: 887-897), phosphoinositides (PIP2 and IP3) and phospholipids (phosphatidic acid) (J. Exptl. Bot., 56: 1665-1674; Protoplasma, 226: 31-38), all modulate  endo/exocytosis  thus supporting our hypothesis of a tight crosstalk in the pollen tube apex.

We further performed a study using a reverse genetics approach and showed that an Arabidopsis lipid kinase, phosphatidylinositol-4-monophosphate 5-kinase 4 (PIP5K4), plays a key role in membrane recycling, cell architecture and polarity of growing pollen tubes (Plant Cell 20: 3050-3064). This enzyme, together with Rab11, seems also to be involved in the asymmetric localization of a specific pollen syntaxin, SYP124 (BMC Plant Biol. 10:179). A similar approach was followed to characterize the role of of FAB1 phosphatidylinositol kinases in Arabidopsis pollen tube growth and fertilization (New Phytol. DOI: 10.1111/nph.12836) and of DAGK4 (New Phytol. doi: 10.1111/nph.15674).

As a result of a collaboration with the group of P. Hussey (Univ of Durham) we published a significant contribution on the regulation of the Arp2/3 complex in plants and how modulation of this complex activity affects the actin cytoskeleton and plant morphogenesis (Curr. Biol. 14: 1410-1414). Further work on actin binding proteins and how their regulation affects microfilaments includes the study of CAP1 (J. Cell Sci., 120: 2609-2618). This Cyclase-Associated Proteins is putatively involved in the link between signalling pathways and structural aspects.

(2) Imaging in Cell Biology

The group is/was also involved in several other projects which involve imaging of cellular structures in live and/or fixed material. Among these are:

a. Imaging of biofilm morphogenesis and mathematical modelling. The main goal of this project was to optimize biofilm growth for industrial purposes. This work was carried out in collaboration with Prof Jonas Almeida (almeida@mail.biol.sc.edu) and Prof M.A. Reis (amr@dq.fct.unl.pt) - Water Science and Technology. 41: 121-127.

b. Imaging of CFTR protein in cystic fibrosis portuguese patients. The aim of this project was to characterize the different mutations present in the portuguese population. This work is carried out in collaboration with Dr Deborah Penque (deborah.Penque@insa.min-saude.pt) and Prof M. Amaral (Chemistry Dept., FCUL) - Laboratory Investigation. 80: 857-868; J. Histochem. & Cytochem., 52: 193-204).

c. Study of ribosomal gene silencing and its effects on chromatin organisation and chromossome structure. This work was carried out in collaboration with Prof Wanda Viegas (wandaviegas@isa.utl.pt) - J. Cell Sci. 115: 2839-2846.

d. Imaging of Ca²⁺ changes in response to cold-shock and cold-acclimation. The aim of this project was to establish the biological reasons underlying the cold-acclimation response in plant cells. In collaboration with Prof Milena Altamura (altamura@uniromal.it) - Plant Sci. 165: 1303-1313. 

e. Study of signal transduction mechanisms in HIV infection and design of intracellular immunization therapies. This work is carried out in collaboration with Prof João Gonçalves (joaogoncalves@ff.ul.pt) - J. Biol. Chem. 277: 32036-32045 and Biotechnology and Applied Biochemistry 59: 353-366.

f. Study of membrane trafficking events and the role of Rab GTPases. This work was carried out in collaboration with Dr Ian Moore, Univ of Oxford (Ian.moore@plants.ox.ac.uk) - Plant Cell 17: 2020-2036.

g. Study of Jasmonate signalling during Organogenesis. This work was carried out in collaboration with Prof M.S. Pais, ICAT-FCUL (maria.pais@fc.ul.pt) - Plant & Cell Physiol. 46: 1713-1723.

h. Evolution of asexual reproduction in leaves of the genus Kalanchoë. This work was carried out in collaboration with Dr Neelima Sinha, Univ of California Davis (nrsinha@ucdavis.edu) - PNAS 104: 15578-155873.

i. Study of the expression of ceruloplasmin and ferroportin in human blood lymphocytes. This work was carried out in collaboration with Prof Luciana Costa (lcosta@insa.min-saude.pt), INSA - Free Radical Biol & Medicine. 44: 483-492.

j. Study of lipid microdomains in Saccharomyces cerevisiae. This work was carried out in collaboration with Prof Luísa Cyrne (mcyrne@fc.ul.pt) and Susana Marinho, CQB-FCUL - Free Radical Biol & Medicine. 46: 289-298.

k. Study of the impact of CdSe/ZnS Quantum Dots in cells of Medicago sativa in suspension culture. This work was carried out in collaboration with Prof Pedro Fevereiro (psalema@itqb.unl.pt), ITQB/FCUL - J. Nanobiotechnology 8:24 

l. The histone deacetylase inhibitor panobinostat is a a promising therapeutic agent for treatment of canine diffuse large B-cell lymphoma. This work was carried out in collaboration with Prof Frederico Aires-da-Silva (fasilva@fmv.ulisboa.pt), FMV - Oncotarget 9: 28586-28598. 

m. Phosphatase and tensin homolog (PTEN) is a growth repressor of both rhizoid and gametophore development in the moss Physcomitrella patens. This work was carried out in collaboration with Prof Laura Saavedra (laura.saavedra@conicet.gov.ar). Plant Physiology 13: 1197- doi.org/10.1104/pp.15.01197