Adverse conditions caused by abiotic stress modulate plant development and growth by altering mor... more Adverse conditions caused by abiotic stress modulate plant development and growth by altering morphological and cellular mechanisms. Plants’ responses/adaptations to stress often involve changes in the distribution and sorting of specific proteins and molecules. Still, little attention has been given to the molecular mechanisms controlling these rearrangements. We tested the hypothesis that plants respond to stress by remodelling their endomembranes and adapting their trafficking pathways. We focused on the molecular machinery behind organelle biogenesis and protein trafficking under abiotic stress conditions, evaluating their effects at the subcellular level, by looking at ultrastructural changes and measuring the expression levels of genes involved in well-known intracellular routes. The results point to a differential response of the endomembrane system, showing that the genes involved in the pathway to the Protein Storage Vacuole and the exocyst-mediated routes are upregulated. ...
In plants, there are several thousands of different types of proteins with different functions th... more In plants, there are several thousands of different types of proteins with different functions that must be correctly located to a specific subcellular compartment. The conventional vacuolar sorting route is already well described and research teams are now more interested in understanding mechanisms behind how unconventional sorting routes work. Our laboratory has been studying the plant-specific insert (PSI), a domain shown to be both sufficient and necessary for correct vacuolar sorting, for a long time. Even though different PSI domains (PSI A and PSI B) present high similarity, they mediate different routes: PSI A has Golgi bypass ability, directly delivering proteins from the endoplasmic reticulum to the vacuole; while PSI B mediates a conventional ER–Golgi–vacuole pathway. The main goal of this study was to identify intermediate players in PSI sorting processes. We purified both PSIs and several endomembrane reporters involved in specific events of protein transport and teste...
Adverse conditions caused by abiotic stress modulate plant development and growth by altering mor... more Adverse conditions caused by abiotic stress modulate plant development and growth by altering morphological and cellular mechanisms. To face this problem, plants, along with physiological adaptations, developed intracellular mechanisms, including changes in protein production and trafficking or modifications of the endomembrane system. It is known that stress situations can alter protein sorting to the vacuole, changing their routes via a Golgi-independent pathway. Our goal is to evaluate the expression levels of different aspartic proteinases and well-characterized genes involved in the vacuolar pathway, in plants submitted to different abiotic stresses (osmotic, oxidative, saline and heavy metals). The results obtained point to a different response of the three aspartic proteinases under study, indicating that different, yet related, genes respond differently to different types of stress, resulting in a fine-tuned regulation. Furthermore, our results regarding the endomembrane sys...
In plant cells, the conventional route to the vacuole involves the endoplasmic reticulum, the Gol... more In plant cells, the conventional route to the vacuole involves the endoplasmic reticulum, the Golgi and the prevacuolar compartment. However, over the years, unconventional sorting to the vacuole, bypassing the Golgi, has been described, which is the case of the Plant-Specific Insert (PSI) of the aspartic proteinase cardosin A. Interestingly, this Golgi-bypass ability is not a characteristic shared by all PSIs, since two related PSIs showed to have different sensitivity to ER-to-Golgi blockage. Given the high sequence similarity between the PSI domains, we sought to depict the differences in terms of post-translational modifications. In fact, one feature that draws our attention is that one is N-glycosylated and the other one is not. Using site-directed mutagenesis to obtain mutated versions of the two PSIs, with and without the glycosylation motif, we observed that altering the glycosylation pattern interferes with the trafficking of the protein as the non-glycosylated PSI-B, unlik...
In plant cells the conventional route to the vacuole involves the endoplasmic reticulum, the Golg... more In plant cells the conventional route to the vacuole involves the endoplasmic reticulum, the Golgi and the prevacuolar compartment. However, over the years, unconventional sorting to the vacuole, bypassing the Golgi, has been described, which is the case of the Plant Specific Insert (PSI) of the aspartic proteinase cardosin A. Interestingly, this Golgi-bypass ability is not a characteristic shared by all PSIs, since two related PSIs showed to have different sensitivity to ER-to-Golgi blockage. Given the high sequence similarity between the PSIs domains, we sought to depict the differences in terms of post-translational modifications. In fact, one feature that draws our attention is that one is N-glycosylated and the other one is not. Using site-directed mutagenesis to obtain mutated versions of the two PSIs, with and without the glycosylation motif, we observed that altering the glycosylation pattern interferes with the trafficking of the protein as the non-glycosylated PSI-B, unlik...
Eukaryotic cells have developed membrane-bound organelles, connected between themselves in a comp... more Eukaryotic cells have developed membrane-bound organelles, connected between themselves in a complex and tightly regulated network – the endomembrane system. Despite being less well understood when compared to the animal and yeast models, plant cells have begun to reveal an intricate and dense network of endomembranes. Particularly diverse is the network of pathways revolving around the vacuole, especially when comparing plant and non-plant models. This dynamic, pleiomorphic and multifunctional organelle is essential for correct plant growth and development, compartmentalizing different components, from proteins to secondary metabolites. In this review we will provide an historical perspective of what has been discovered relating vacuolar sorting, and the potential biotech applications of such findings.
Thesis Abstract The process of tracheary elements differentiation and cell metabolism were examin... more Thesis Abstract The process of tracheary elements differentiation and cell metabolism were examined in an established callus tissue derived from leaf mesophyll of Sedum telephium L. currently maintained on Gamborg's B5 medium. 1. The ultrastructure of differentiating sieve elements and xylem tracheary elements was also studied in the leaf of S. telephium. The ultrastructural aspects observed throughout the process of differentiation of the parenchyma cells, phloem and xylem elements are in accordance with the descriptions found on the literature about this matter. During the development of the leaf, parenchyma cells, inicially small, with thin walls and without intercellular spaces, resumed meristematic activity and divided and enlarged following predicted patterns. In both cell division and cell polarity is ascribed to citoskeleton. 1.1. Sieve elements at early stages of differentiation resemble meristematic cells. The ultrastructural features of differentiating phloem elements...
Dissertação de Doutoramento em Biologia, área de especialização em Anatomia Vegetal, apresentada ... more Dissertação de Doutoramento em Biologia, área de especialização em Anatomia Vegetal, apresentada à Faculdade de Ciências da Universidade do Porto
The relationship between growth, chlorophylls, soluble proteins, carbohydrates, and phenolic comp... more The relationship between growth, chlorophylls, soluble proteins, carbohydrates, and phenolic compounds in a callus culture of Sedum telephium grown for 20 days on three culture media each with different nitrogen contents was studied. Growing the callus tissue in B5 medium containing 24.70 mM nitrate plus 2.0 mM ammonium as the nitrogen source, resulted in a typical sigmoidal growth curve and induced early carbohydrate breakdown; this rate of carbohydrate utilization was correlated with an increased rate of protein synthesis and reduced phenolic production and accumulation. Omitting ammonium from the culture medium (leaving 24.70 mM nitrate as the only nitrogen source) decreased growth rate of the tissue but induced early formation of soluble phenolics, with no effect on chlorophyll levels. A further decrease in nitrogen content by reducing the nitrate concentration, from 24.70 to 12.35 mM (with no ammonium), reduced chlorophyll levels and delayed carbohydrate breakdown. The soluble ...
The 18th century Portuguese church furniture represents an extraordinary richness recognised worl... more The 18th century Portuguese church furniture represents an extraordinary richness recognised worldwide, which demands safeguarding and valorisation. The identification of the wood of furniture artworks is the most important component for its comprehension and preservation. In this work wood anatomical characters of an 18th century Portuguese decorative furniture set from the Colegiada de São Martinho de Cedofeita, in Porto, were analysed to identify the woods used for manufacturing and to clarify their common names. Furthermore, the objectives were to recognise some of the criteria for choice of wood as well as the source of each wood. The woods identified from 16 fragments belong to Apuleia sp., Acacia sp., Neolamarckia sp. and Castanea sativa. Apuleia sp. and Acacia sp. woods most likely arrived from Brazil, while the Neolamarckia sp. woods likely arrived from India and the C. sativa woods from Portugal. The results are in accordance with the known Portuguese colonial sea routes o...
We studied decorative Portuguese furniture from the 18th century Clérigos Church in Porto, to ide... more We studied decorative Portuguese furniture from the 18th century Clérigos Church in Porto, to identify the woods used, to analyze their possible origin and understand the criteria for wood choice, according to sample location. We identified wood from Acacia sp., Castanea sativa, Couratari sp. and Dalbergia nigra. D. nigra, Acacia sp. and Couratari sp. may have arrived from Brazil, according to their natural distribution and the Portuguese colonial routes; C. sativa was abundant in the North Region of Portugal. In the 18th century, golden metal brass over black furniture was in fashion in Portuguese ecclesiastical cabinet making. Due to its dark colour, Dalbergia nigra was a desirable wood for the exposed structures of ecclesiastical furniture, such as top drawers and top tables. Couratari is a pale wood. Therefore, it was used in the inner structure of the drawers and legs. Acacia wood was also used in internal parts due to its durability. C. sativa was local and extensive; its wood...
Our environment is contaminated with organic and inorganic compounds released by anthropogenic ac... more Our environment is contaminated with organic and inorganic compounds released by anthropogenic activities that cause negative impacts on biological productivity and ecosystem sustainability and place human health at risk. Within the available remediation technologies, phytoremediation has emerged with high potential due to its reduced environmental impacts and economic costs. The research into phytoremediation has developed through a wide array of approaches, which also pertains to its inherent interdisciplinary characteristics, towards enhancing the potential of the technology for application in the field. Numerous patents present molecular solutions through which plants can be engineered to display improvements in key characteristics, such as the tolerance, uptake and accumulation of contaminants. The manipulation of plant growth and of the physico-chemical characteristics of the contaminated environments in order to enhance the remediation potential has also been the focus of several issued patents. This review attempts to highlight the most relevant patented advances in phytoremediation and to emphasise recent research efforts through which this green technology might be expected to develop into a commercially competitive alternative to other remediation methods.
Zinc tolerance, accumulation, and organic acid production by Solanum nigrum, a known Zn accumulat... more Zinc tolerance, accumulation, and organic acid production by Solanum nigrum, a known Zn accumulator, was studied during pre- and post-flowering stages of development. The plants, when challenged with Zn concentrations lethal to plantlets, showed an increase in tolerance from pre-flowering to post-flowering, which was accompanied by a reduction of Zn translocation to the aerial plant parts. Treatment with Zn induced a differential response in organic acids according to the plant organ and developmental stage. In the roots, where Zn concentrations were similar in pre- and post-flowering plants, a general decrease in organic acid in pre-flowering roots contrasted with the increase observed in post-flowering plants. In the stems, Zn induced a generalized increase in organic acids at both growth stages while in the leaves, a slight increase in malic and shikimic was observed in pre-flowering plants and only shikimic acid levels were significantly increased in post-flowering plants. This work shows that Zn accumulation and tolerance in S. nigrum vary during plant development - an observation that may be important to improve the efficiency of phytoremediation approaches. Furthermore, the data suggest the involvement of specific organic acids in this response.
Advances in Experimental Medicine and Biology, 1998
ABSTRACT A feature common to many proteases, including aspartic proteinases, is that they are syn... more ABSTRACT A feature common to many proteases, including aspartic proteinases, is that they are synthesised as inactive zymogens, subsequently undergoing proteolytic processing to yield the active enzyme. This is a way of assuring the correct folding of the proteinase, regulating its activity during biosynthesis and avoiding unwanted proteolysis. Most aspartic proteinases have a conserved N-terminal pro-segment, which is later removed (1). In pepsinogen, the pro-segment is located over the active site cleft, stabilized by salt bridges that are disrupted at low pH (2). In cathepsin D, apart from the N-terminal pro-segment and a C-terminal dipeptide there is another sequence which is also removed, located within the protein (3). Removal of this 2–7 amino acid long sequence gives rise to the two chain active cathepsin D.
Adverse conditions caused by abiotic stress modulate plant development and growth by altering mor... more Adverse conditions caused by abiotic stress modulate plant development and growth by altering morphological and cellular mechanisms. Plants’ responses/adaptations to stress often involve changes in the distribution and sorting of specific proteins and molecules. Still, little attention has been given to the molecular mechanisms controlling these rearrangements. We tested the hypothesis that plants respond to stress by remodelling their endomembranes and adapting their trafficking pathways. We focused on the molecular machinery behind organelle biogenesis and protein trafficking under abiotic stress conditions, evaluating their effects at the subcellular level, by looking at ultrastructural changes and measuring the expression levels of genes involved in well-known intracellular routes. The results point to a differential response of the endomembrane system, showing that the genes involved in the pathway to the Protein Storage Vacuole and the exocyst-mediated routes are upregulated. ...
In plants, there are several thousands of different types of proteins with different functions th... more In plants, there are several thousands of different types of proteins with different functions that must be correctly located to a specific subcellular compartment. The conventional vacuolar sorting route is already well described and research teams are now more interested in understanding mechanisms behind how unconventional sorting routes work. Our laboratory has been studying the plant-specific insert (PSI), a domain shown to be both sufficient and necessary for correct vacuolar sorting, for a long time. Even though different PSI domains (PSI A and PSI B) present high similarity, they mediate different routes: PSI A has Golgi bypass ability, directly delivering proteins from the endoplasmic reticulum to the vacuole; while PSI B mediates a conventional ER–Golgi–vacuole pathway. The main goal of this study was to identify intermediate players in PSI sorting processes. We purified both PSIs and several endomembrane reporters involved in specific events of protein transport and teste...
Adverse conditions caused by abiotic stress modulate plant development and growth by altering mor... more Adverse conditions caused by abiotic stress modulate plant development and growth by altering morphological and cellular mechanisms. To face this problem, plants, along with physiological adaptations, developed intracellular mechanisms, including changes in protein production and trafficking or modifications of the endomembrane system. It is known that stress situations can alter protein sorting to the vacuole, changing their routes via a Golgi-independent pathway. Our goal is to evaluate the expression levels of different aspartic proteinases and well-characterized genes involved in the vacuolar pathway, in plants submitted to different abiotic stresses (osmotic, oxidative, saline and heavy metals). The results obtained point to a different response of the three aspartic proteinases under study, indicating that different, yet related, genes respond differently to different types of stress, resulting in a fine-tuned regulation. Furthermore, our results regarding the endomembrane sys...
In plant cells, the conventional route to the vacuole involves the endoplasmic reticulum, the Gol... more In plant cells, the conventional route to the vacuole involves the endoplasmic reticulum, the Golgi and the prevacuolar compartment. However, over the years, unconventional sorting to the vacuole, bypassing the Golgi, has been described, which is the case of the Plant-Specific Insert (PSI) of the aspartic proteinase cardosin A. Interestingly, this Golgi-bypass ability is not a characteristic shared by all PSIs, since two related PSIs showed to have different sensitivity to ER-to-Golgi blockage. Given the high sequence similarity between the PSI domains, we sought to depict the differences in terms of post-translational modifications. In fact, one feature that draws our attention is that one is N-glycosylated and the other one is not. Using site-directed mutagenesis to obtain mutated versions of the two PSIs, with and without the glycosylation motif, we observed that altering the glycosylation pattern interferes with the trafficking of the protein as the non-glycosylated PSI-B, unlik...
In plant cells the conventional route to the vacuole involves the endoplasmic reticulum, the Golg... more In plant cells the conventional route to the vacuole involves the endoplasmic reticulum, the Golgi and the prevacuolar compartment. However, over the years, unconventional sorting to the vacuole, bypassing the Golgi, has been described, which is the case of the Plant Specific Insert (PSI) of the aspartic proteinase cardosin A. Interestingly, this Golgi-bypass ability is not a characteristic shared by all PSIs, since two related PSIs showed to have different sensitivity to ER-to-Golgi blockage. Given the high sequence similarity between the PSIs domains, we sought to depict the differences in terms of post-translational modifications. In fact, one feature that draws our attention is that one is N-glycosylated and the other one is not. Using site-directed mutagenesis to obtain mutated versions of the two PSIs, with and without the glycosylation motif, we observed that altering the glycosylation pattern interferes with the trafficking of the protein as the non-glycosylated PSI-B, unlik...
Eukaryotic cells have developed membrane-bound organelles, connected between themselves in a comp... more Eukaryotic cells have developed membrane-bound organelles, connected between themselves in a complex and tightly regulated network – the endomembrane system. Despite being less well understood when compared to the animal and yeast models, plant cells have begun to reveal an intricate and dense network of endomembranes. Particularly diverse is the network of pathways revolving around the vacuole, especially when comparing plant and non-plant models. This dynamic, pleiomorphic and multifunctional organelle is essential for correct plant growth and development, compartmentalizing different components, from proteins to secondary metabolites. In this review we will provide an historical perspective of what has been discovered relating vacuolar sorting, and the potential biotech applications of such findings.
Thesis Abstract The process of tracheary elements differentiation and cell metabolism were examin... more Thesis Abstract The process of tracheary elements differentiation and cell metabolism were examined in an established callus tissue derived from leaf mesophyll of Sedum telephium L. currently maintained on Gamborg's B5 medium. 1. The ultrastructure of differentiating sieve elements and xylem tracheary elements was also studied in the leaf of S. telephium. The ultrastructural aspects observed throughout the process of differentiation of the parenchyma cells, phloem and xylem elements are in accordance with the descriptions found on the literature about this matter. During the development of the leaf, parenchyma cells, inicially small, with thin walls and without intercellular spaces, resumed meristematic activity and divided and enlarged following predicted patterns. In both cell division and cell polarity is ascribed to citoskeleton. 1.1. Sieve elements at early stages of differentiation resemble meristematic cells. The ultrastructural features of differentiating phloem elements...
Dissertação de Doutoramento em Biologia, área de especialização em Anatomia Vegetal, apresentada ... more Dissertação de Doutoramento em Biologia, área de especialização em Anatomia Vegetal, apresentada à Faculdade de Ciências da Universidade do Porto
The relationship between growth, chlorophylls, soluble proteins, carbohydrates, and phenolic comp... more The relationship between growth, chlorophylls, soluble proteins, carbohydrates, and phenolic compounds in a callus culture of Sedum telephium grown for 20 days on three culture media each with different nitrogen contents was studied. Growing the callus tissue in B5 medium containing 24.70 mM nitrate plus 2.0 mM ammonium as the nitrogen source, resulted in a typical sigmoidal growth curve and induced early carbohydrate breakdown; this rate of carbohydrate utilization was correlated with an increased rate of protein synthesis and reduced phenolic production and accumulation. Omitting ammonium from the culture medium (leaving 24.70 mM nitrate as the only nitrogen source) decreased growth rate of the tissue but induced early formation of soluble phenolics, with no effect on chlorophyll levels. A further decrease in nitrogen content by reducing the nitrate concentration, from 24.70 to 12.35 mM (with no ammonium), reduced chlorophyll levels and delayed carbohydrate breakdown. The soluble ...
The 18th century Portuguese church furniture represents an extraordinary richness recognised worl... more The 18th century Portuguese church furniture represents an extraordinary richness recognised worldwide, which demands safeguarding and valorisation. The identification of the wood of furniture artworks is the most important component for its comprehension and preservation. In this work wood anatomical characters of an 18th century Portuguese decorative furniture set from the Colegiada de São Martinho de Cedofeita, in Porto, were analysed to identify the woods used for manufacturing and to clarify their common names. Furthermore, the objectives were to recognise some of the criteria for choice of wood as well as the source of each wood. The woods identified from 16 fragments belong to Apuleia sp., Acacia sp., Neolamarckia sp. and Castanea sativa. Apuleia sp. and Acacia sp. woods most likely arrived from Brazil, while the Neolamarckia sp. woods likely arrived from India and the C. sativa woods from Portugal. The results are in accordance with the known Portuguese colonial sea routes o...
We studied decorative Portuguese furniture from the 18th century Clérigos Church in Porto, to ide... more We studied decorative Portuguese furniture from the 18th century Clérigos Church in Porto, to identify the woods used, to analyze their possible origin and understand the criteria for wood choice, according to sample location. We identified wood from Acacia sp., Castanea sativa, Couratari sp. and Dalbergia nigra. D. nigra, Acacia sp. and Couratari sp. may have arrived from Brazil, according to their natural distribution and the Portuguese colonial routes; C. sativa was abundant in the North Region of Portugal. In the 18th century, golden metal brass over black furniture was in fashion in Portuguese ecclesiastical cabinet making. Due to its dark colour, Dalbergia nigra was a desirable wood for the exposed structures of ecclesiastical furniture, such as top drawers and top tables. Couratari is a pale wood. Therefore, it was used in the inner structure of the drawers and legs. Acacia wood was also used in internal parts due to its durability. C. sativa was local and extensive; its wood...
Our environment is contaminated with organic and inorganic compounds released by anthropogenic ac... more Our environment is contaminated with organic and inorganic compounds released by anthropogenic activities that cause negative impacts on biological productivity and ecosystem sustainability and place human health at risk. Within the available remediation technologies, phytoremediation has emerged with high potential due to its reduced environmental impacts and economic costs. The research into phytoremediation has developed through a wide array of approaches, which also pertains to its inherent interdisciplinary characteristics, towards enhancing the potential of the technology for application in the field. Numerous patents present molecular solutions through which plants can be engineered to display improvements in key characteristics, such as the tolerance, uptake and accumulation of contaminants. The manipulation of plant growth and of the physico-chemical characteristics of the contaminated environments in order to enhance the remediation potential has also been the focus of several issued patents. This review attempts to highlight the most relevant patented advances in phytoremediation and to emphasise recent research efforts through which this green technology might be expected to develop into a commercially competitive alternative to other remediation methods.
Zinc tolerance, accumulation, and organic acid production by Solanum nigrum, a known Zn accumulat... more Zinc tolerance, accumulation, and organic acid production by Solanum nigrum, a known Zn accumulator, was studied during pre- and post-flowering stages of development. The plants, when challenged with Zn concentrations lethal to plantlets, showed an increase in tolerance from pre-flowering to post-flowering, which was accompanied by a reduction of Zn translocation to the aerial plant parts. Treatment with Zn induced a differential response in organic acids according to the plant organ and developmental stage. In the roots, where Zn concentrations were similar in pre- and post-flowering plants, a general decrease in organic acid in pre-flowering roots contrasted with the increase observed in post-flowering plants. In the stems, Zn induced a generalized increase in organic acids at both growth stages while in the leaves, a slight increase in malic and shikimic was observed in pre-flowering plants and only shikimic acid levels were significantly increased in post-flowering plants. This work shows that Zn accumulation and tolerance in S. nigrum vary during plant development - an observation that may be important to improve the efficiency of phytoremediation approaches. Furthermore, the data suggest the involvement of specific organic acids in this response.
Advances in Experimental Medicine and Biology, 1998
ABSTRACT A feature common to many proteases, including aspartic proteinases, is that they are syn... more ABSTRACT A feature common to many proteases, including aspartic proteinases, is that they are synthesised as inactive zymogens, subsequently undergoing proteolytic processing to yield the active enzyme. This is a way of assuring the correct folding of the proteinase, regulating its activity during biosynthesis and avoiding unwanted proteolysis. Most aspartic proteinases have a conserved N-terminal pro-segment, which is later removed (1). In pepsinogen, the pro-segment is located over the active site cleft, stabilized by salt bridges that are disrupted at low pH (2). In cathepsin D, apart from the N-terminal pro-segment and a C-terminal dipeptide there is another sequence which is also removed, located within the protein (3). Removal of this 2–7 amino acid long sequence gives rise to the two chain active cathepsin D.
Eukaryotic cells have developed membrane-bound organelles, connected between themselves in a comp... more Eukaryotic cells have developed membrane-bound organelles, connected between themselves in a complex and tightly regulated network – the endomembrane system. Despite being less well understood when compared to the animal and yeast models, plant cells have begun to reveal an intricate and dense network of endomembranes. Particularly diverse is the network of pathways revolving around the vacuole, especially when comparing plant and non-plant models. This dynamic, pleiomorphic and multifunctional organelle is essential for correct plant growth and development, compartmentalizing different components, from proteins to secondary metabolites. In this review we will provide an historical perspective of what has been discovered relating vacuolar sorting, and the potential biotech applications of such findings.
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