Biologia Molecular Vegetal

Interesse da Investigação

The Plant Molecular Biology lab uses Arabidopsis thaliana as a model system to investigate how plants perceive and respond to environmental signals and endogenous developmental cues at the molecular level. In particular, we are focusing on the role of pre-mRNA splicing in the regulation of gene expression. The versatility of this mode of regulation suggests that it is likely to play an important contribution in ensuring the developmental plasticity and stress tolerance that are essential for plant survival. Another major ongoing project in the lab is examining the role of mutiple drug resistance (MDR) transporters in plant resistance to pesticides and other chemical stresses.

Membros do Grupo

Projecto de Investigação

Alternative Splicing in Plant Development and Stress Response

Alternative splicing has recently emerged as one of the most important mechanisms for generating proteome diversity and regulating gene expression. It is becoming clear that the majority of human genes encode transcripts that undergo alternative splicing, greatly enhancing the coding potential of the genome. Few examples of alternatively-spliced transcripts have been reported in plants and still fewer are known to generate functionally distinct proteins. However, the recent explosive growth of available expressed sequence data and its alignment with genomic sequences indicates that alternative splicing plays a far more important role in plants than previously thought, and further work is necessary to uncover the significance and regulation of this process. The unique developmental plasticity and stress tolerance developed by plants as a result of their sessile growth habit suggests that they offer exceptional opportunities to reveal alternative splicing mechanisms, which are likely to play a key role in the adaptation of plants to changes in their environment. We are investigating both the biological relevance of alternative splicing in plant development and stress responses and the regulatory mechanisms of plant pre-mRNA splicing about which virtually nothing is known. The functional significance of different splice variants of specific development- and stress-associated genes is being examined using reverse genetic approaches in Arabidopsis thaliana. Moreover, the function of individual serine/arginine-rich (SR) proteins, which are established key players in mammalian alternative splicing, is being analyzed. This work deals with a poorly explored area of plant biology and may accelerate the understanding of the molecular mechanisms underlying plant adaptation to environmental stress, opening new avenues for the use of biotechnology to increase plant productivity.

Funding

POCI/DG/BIA/82009/2006
Alternative Splicing in Plant Development and Stress Response

Projecto de Investigação

Herbicide resistance in Arabidopsis thaliana: Role of plant multidrug resistance transporters

The widespread use of herbicides has led to an increasing number of resistant weed species and classes of herbicides to which resistance has evolved, with some biotypes currently showing multiple-resistance to various of these agrochemicals. Herbicide resistance has the potential to cause not only large economic losses in agriculture, but also deleterious effects on the environment and human health, as a result of rising herbicide application rates. The lack of a basic understanding of the molecular mechanisms underlying herbicide resistance remains the greatest obstacle to the use of biotechnology to deal with this problem. The Biological Sciences Research Group at Instituto Superior Técnico (IST) has recently shown that three Saccharomyces cerevisiae genes encoding plasma membrane multiple drug resistance (MDR) transporters determine yeast resistance to 2,4-dichlorophenoxyacetic acid (2,4-D), one of the selective herbicides most successfully used worldwide. Two of these genes, ScPDR5 and ScPDR18, belong to the pleiotropic drug resistance (PDR) subfamily of ABC drug efflux pumps, whereas the other MDR transporter (ScTPO1) is a member of the major facilitator superfamily (MFS). Based on these findings, we have been analyzing the effects of 2,4-D application on the expression of poorly characterized Arabidopsis thaliana MDR transporter genes showing high sequence similarity to the yeast resistance determinants. First results show a clear transcriptional activation of one MFS and two PDR genes in response to the herbicide. This strongly suggests an involvement of these putative plasma membrane drug efflux pumps in the plant’s response to herbicidal concentrations of 2,4-D and pinpoints excellent candidates for herbicide resistance determinants in A. thaliana. The IGC and IST groups are collaborating to investigate the role of genes encoding MDR transporters of the ABC and MF Superfamilies in plant herbicide resistance. The functional analysis of these novel or poorly characterized plant genes involve gene expression analysis and reverse genetic approaches in Arabidopsis, heterologous expression and complementation experiments in yeast, and membrane transport assays in both systems. These studies, using the plant model A. thaliana and the fundamental model eukaryote S. cerevisiae, should accelerate understanding of the molecular mechanisms governing herbicide resistance, currently one of the most unexplored and exciting topics in plant biology, with important implications for agriculture, the environment, and human health.

Funding

PTDC/AGR-AAM/67858/2006
Herbicide resistance in Arabidopsis thaliana: Role of plant multidrug resistance transporters

Colaboradores

Instituto Superior Técnico
Isabel Sá-Correia

Projecto de Investigação

Alternative Splicing of an Arabidopsis RING E3 ubiquitin ligase involved in plant stress and development

As sessile organisms, plants developed adaptive developmental and physiological strategies to cope with environmental stress. These range from morphological modifications to physiological adaptation at the cellular level, but the basis of the capacity for adaptation lies ultimately at the level of the genome. Alternative splicing has recently emerged as an important mechanism for generating proteome diversity and regulating gene expression. Its prevalence in many genomes shows that it may play important roles in biological processes, and recent studies indicate that plant alternative splicing events occur in response to environmental changes or at particular developmental stages. Nevertheless, few alternatively-spliced transcripts have been reported in plants so far. On the other hand, the ubiquitin/26S proteasome pathway, a tightly regulated and highly specific protein degradation system essential for organism survival, contributes significantly to plant development and stress tolerance by affecting a wide range of processes, and several plant E3 ubiquitin ligases have been implicated in the transduction of environmental signals. In preliminary studies, we have identified an Arabidopsis thaliana gene encoding a RING E3 ligase involved in protein ubiquitination that undergoes alternative splicing. Exon skipping excludes the only nuclear localization signal in the predicted protein and determines the subcellular localization of two splice forms — the longer isoform is targeted to the nucleus, while the shorter localizes to the cytoplasm. Both alternatively-spliced transcripts are upregulated during senescence and upon application of the stress- response hormone abscisic acid (ABA), but the magnitude of this induction is more than two fold higher for the splice variant encoding the nuclear isoform. These results strongly suggest a role for alternative splicing of this ubiquitination gene in plant developmental control and stress responses. We have been extending these studies and conducting a detailed functional analysis of this poorly characterized RING E3 ligase. To that end, stress- and development-specific expression and splicing patterns are being investigated and reverse genetics approaches in Arabidopsis used to examine the functional significance of both splice forms. Moreover, assessment of the ubiquitination capacity of each isoform and identification of the target protein(s) using in vitro ubiquitination and yeast two-hybrid assays hold much promise for unraveling the mode of action of this alternatively-spliced gene and its role in plant development and stress.

Funding

PTDC/AGR/GPL/70345/2006
Alternative Splicing of an Arabidopsis RING E3 ubiquitin ligase involved in plant stress and development

Publicações

Duque, P. (2011). A role for SR proteins in plant stress responses Plant Signal. Behav. 6 :49-54

Cabrito T.R., Remy E., Teixeira M.C., Duque P., Sá-Correia I. (2011). Resistance to herbicides in the model organisms Saccharomyces cerevisiae and Arabidopsis thaliana: the involvement of multidrug resistance transporters Herbicides and Environment (ed A. Kortekamp), InTech, Vienna, Austria :623-640

Carvalho, R.F., Carvalho, S.D., Duque, P. (2010). The plant-specific SR45 protein negatively regulates glucose and ABA signaling during early seedling development in Arabidopsis Plant Physiol 154 :772-783

Carvalho, I.S., Cavaco, T., Carvalho, L.M., Duque, P. (2010). Effect of photoperiod on flavonoid pathway activity in sweet potato (Ipomoea batatas (L.) Lam.) leaves Food Chem 118 :384-390

Cabrito, T.R., Teixeira, M.C., Duarte, A.A., Duque, P., Sá-Correia, I. (2009). Heterologous expression of a TPO1 homolog from Arabidopsis thaliana confers resistance to the herbicide 2,4-D and other chemical stresses in yeast. Appl Microbiol Biotechnol 84 :927-936

Teixeira, M.C., Duque, P., Sá-Correia, I. (2007). Environmental Genomics: mechanistic insights into toxicity and resistance to the herbicide 2,4-D Trends Biotech 258 :363-370

Kameoka, S.*, Duque, P.*, Konarska, M.M. (2004). p54nrb associates with the 5’ splice site within large transcription/splicing complexes. EMBO J. 23 :1782-1791

Duque, P., Sánchez, J.P., Chua, N.-H. (2004). Signaling and the cytoskeleton in guard cells, in The Plant Cytoskeleton in Cell Differentiation and Development (ed P.J. Hussey) Blackwell Publishing, Oxford, U.K., pp :290-317

Sánchez, J.P., Duque, P., Chua, N.-H. (2004). ABA activates ADPR cyclase and cADPR induces a subset of ABA-responsive genes in Arabidopsis. Plant J. 38 :381-95

Duque, P., Chua, N.-H. (2003). IMB1, a bromodomain protein induced during seed imbibition, regulates ABA- and phyA-mediated responses of germination in Arabidopsis. Plant J. 35 :787-799

Duque, P., Barreiro, M.G., Arrabaça, J.D. (1999). Respiratory metabolism during cold storage of apple fruit. I. Sucrose metabolism and glycolysis. Physiol. Plant. 107 :14-23

Duque, P., Arrabaça, J.D. (1999). Respiratory metabolism during cold storage of apple fruit. II. Alternative oxidase is induced at the climacteric. Physiol. Plant. 107 :24-31