Leonor Boavida - Academia.edu (original) (raw)

Papers by Leonor Boavida

Journal of Experimental Botany, 2010

The process of pollen germination and tube growth in the pistil involves a series of cell-cell in... more The process of pollen germination and tube growth in the pistil involves a series of cell-cell interactions, some facilitating fertilization while others prohibiting pollen tube access to the female gametophyte, either because of incompatibility or as a result of mechanisms to avert polyspermy and to ensure reproductive success. Understanding pollen tube growth and guidance to the female gametophyte has long been a pursuit among plant biologists, and observations indicate that diverse strategies may be adopted by different plant species. Recent studies in Arabidopsis, maize, and Torenia fournieri suggest that low molecular weight secretory molecules probably play major roles in the short-range attraction of pollen tubes to the female gametophyte. The process of pollen tube growth in the pistil occurs beneath several cell layers so much of the information that conveys the intimate partnership between penetrating pollen tubes and the female tissues has come from fixed samples and observations of in vitro pollen tube growth responses to female factors. A unique glimpse of the in vivo pollen germination and tube growth process is provided here by intra-vital two-photon excitation (TPE) microscopy of pollinated Arabidopsis pistils that remained on intact plants. Further discoveries of critical factors of male or female origins and how they control the pollen tube growth and fertilization process will broaden our understanding of the common themes and diverse strategies that plants have evolved to ensure reproductive success. The advancement of imaging technology to monitor pollination and fertilization and the development of probes to monitor various aspects of the pollen tube growth process, including pollen intracellular dynamics, will allow us to superimpose details obtained from studying pollen tube growth in culture conditions to interpret and understand the in vivo events.

Sexual Plant Reproduction, 2001

Intraspecific barriers promote outcrossing while interspecific mechanisms may contribute to the i... more Intraspecific barriers promote outcrossing while interspecific mechanisms may contribute to the isolation of species; both control the exchange of genes between plants. In this paper we establish that post-pollination mechanisms promote outcross and act at different temporal and spatial levels to control seed set and quality in Quercus species. Controlled pollinations were performed to investigate intraspecific crossing barriers in Q. suber and pollen-pistil interactions following interspecific pollinations with some simpatric Quercus species. Cytological data of intraspecific crosses in Q. suber and Q. ilex have shown different kinetics on pollen tube growth after self and outcross pollination, but pollen tubes were able to reach the base of the styles in both species and seed set was successful. Although some pre-zygotic interaction is occurring at the style, the most important interaction takes place at ovary. The cross between Q. ilex and Q. suber is possible only in one direction, revealing a case of unilateral incongruity. We show that the lack of seed set observed in the cross Q. suber×Q. ilex is due to the inability of pollen tubes to penetrate the transmitting tissue after germination. With Q. suber mainly as female parent, pollinations with other simpatric Quercus species have shown different levels of constraint on pollen tube progression at stigma and style levels. Eventual hybridisation between Quercus species will depend on the compatibility of pollen-pistil interactions, on the competitive ability of pollen genotypes, and, most important, on the overlapping of geographical, phenological and ecological factors. Differences in seed set and seed allocation was evident in all crosses, and was particularly outstanding in interspecific and in self intraspecific crosses, determining the ultimate level of seed production and quality in Quercus species.

Plant Cell, 2008

Pollen tubes must navigate through different female tissues to deliver sperm to the embryo sac fo... more Pollen tubes must navigate through different female tissues to deliver sperm to the embryo sac for fertilization. Protein disulfide isomerases play important roles in the maturation of secreted or plasma membrane proteins. Here, we show that certain T-DNA insertions in Arabidopsis thaliana PDIL2-1, a protein disulfide isomerase (PDI), have reduced seed set, due to delays in embryo sac maturation. Reciprocal crosses indicate that these mutations acted sporophytically, and aniline blue staining and scanning electron microscopy showed that funicular and micropylar pollen tube guidance were disrupted. A PDIL2-1-yellow fluorescent protein fusion was mainly localized in the endoplasmic reticulum and was expressed in all tissues examined. In ovules, expression in integument tissues was much higher in the micropylar region in later developmental stages, but there was no expression in embryo sacs. We show that reduced seed set occurred when another copy of full-length PDIL2-1 or when enzymatically active truncated versions were expressed, but not when an enzymatically inactive version was expressed, indicating that these T-DNA insertion lines are gain-of-function mutants. Our results suggest that these truncated versions of PDIL2-1 function in sporophytic tissues to affect ovule structure and impede embryo sac development, thereby disrupting pollen tube guidance.

Plant Journal, 2007

Despite much effort, a robust protocol for in vitro germination of Arabidopsis thaliana pollen ha... more Despite much effort, a robust protocol for in vitro germination of Arabidopsis thaliana pollen has been elusive. Here we show that controlled temperatures, a largely disregarded factor in previous studies, and a simple optimized medium, solidified or liquid, yielded pollen germination rates above 80% and pollen tube lengths of hundreds of microns, with both Columbia and Landsberg erecta (Ler) ecotypes. We found that pollen germination and tube growth were dependent on pollen density in both liquid and solid medium. Pollen germination rates were not substantially affected by flower or plant age. The quartet1 mutation negatively affected pollen germination, especially in the Ler ecotype. This protocol will facilitate functional analyses of insertional mutants affecting male gametophyte function, and should allow detailed gene expression analyses during pollen tube growth. Arabidopsis thaliana can now be included on the list of plant species that are suitable models for physiological studies of pollen tube elongation and tip growth.

Genetics, 2009

Functional analyses of the Arabidopsis genome require analysis of the gametophytic generation, si... more Functional analyses of the Arabidopsis genome require analysis of the gametophytic generation, since about 10% of the genes are expressed in the male gametophyte and about 9% in the female gametophyte. Here we describe the genetic and molecular characterization of 67 Ds insertion lines that show reduced transmission through the male gametophyte. About half of these mutations are male gametophytic-specific mutations, while the others also affect female transmission. Genomic sequences flanking both sides of the Ds element were recovered for 39 lines; for 16 the Ds elements were inserted in or close to coding regions, while 7 were located in intergenic/unannotated regions of the genome. For the remaining 16 lines, chromosomal rearrangements such as translocations or deletions, ranging between 30 kb to 500 kb, were associated with the transposition event. The mutants were classified into five groups according to the developmental processes affected; these ranged from defects in early stages of gametogenesis to later defects affecting pollen germination, pollen tube growth, polarity or guidance, or pollen tube-embryo sac interactions or fertilization. The isolated mutants carry Ds insertions in genes with diverse biological functions and potentially specify new functions for several unannotated or unknown proteins.

International Journal of Developmental Biology, 2005

The evolutionary success of higher plants relies on a very short gametophytic phase, which underl... more The evolutionary success of higher plants relies on a very short gametophytic phase, which underlies the sexual reproduction cycle. Sexual plant reproduction takes place in special organs of the flower: pollen, the male gametophyte, is released from the anthers and then adheres, grows and interacts along various tissues of the female organs, collectively known as the pistil. Finally, it fertilizes the female gametophyte, the embryo sac. Pollen is released as bi or tricellular, highly de-hydrated and presumably containing all the biochemical components and transcripts to germinate. Upon hydration on the female tissues, it develops a cytoplasmic extension, the pollen tube, which is one of the fastest growing cells in nature. Pollen is completely "ready-to-go", but despite this seemingly simple reaction, very complex interactions take place with the female tissues. In higher animals, genetic mechanisms for sex determination establish striking developmental differences between males and females. In contrast, most higher plant species develop both male and female structures within the same flower, allowing self-fertilization. Outcrossing is ensured by self-incompatibility mechanisms, which evolved under precise genetic control, controlling self-recognition and cell-to-cell interaction. Equally important is pollen selection along the female tissues, where interactions between different cell types with inherent signalling properties correspond to check-points to ensure fertilization. Last but not least, pollen-pistil interaction occurs in a way that enables the correct targeting of the pollen tubes to the receptive ovules. In this review, we cover the basic mechanisms underlying sexual plant reproduction, from the structural and cellular determinants, to the most recent genetic advances.

International Journal of Developmental Biology, 2005

Higher plants have evolved to be one of the predominant life forms on this planet. A great deal o... more Higher plants have evolved to be one of the predominant life forms on this planet. A great deal of this evolutionary success relies in a very short gametophytic phase which underlies the sexual reproduction cycle. Sexual plant reproduction takes place in special organs of the flower. In most species the processes of gametogenesis, pollination, syngamy and embryogenesis are sequentially coordinated to give rise to a functional seed in a matter of few weeks. Any of these processes is so intricately complex and precisely regulated that it becomes no wonder that each involves more specific genes and cellular processes than any other function in the plant life cycle. While variability generation -the evolutionary output of the sexual cycle -is the same as in any other Kingdom, plants do it using a completely original set of mechanisms, many of which are not yet comprehended. In this paper, we cover the fundamental features of male and female gametogenesis. While the physiological and cellular bases of these processes have been continuously described since the early nineteen century, recent usage of Arabidopsis and other species as central models has brought about a great deal of specific information regarding their genetic regulation. Transcriptomics has recently enlarged the repertoire and pollen became the first gametophyte to have a fully described transcriptome in plants. We thus place special emphasis on the way this newly accumulated genetic and transcriptional information impacts our current understanding of the mechanisms of gametogenesis.

Sexual Plant Reproduction, 1999

Cork oak (Quercus suber L.) is a monoecious wind-pollinated species with a protandrous system to... more Cork oak (Quercus suber L.) is a monoecious wind-pollinated species with a protandrous system to ensure cross-pollination. To the best of our knowledge, this report provides the first insight into the sexual reproduction cycle in this species. The cork oak flowering season extends from April until the end of May. Our results show that, at anthesis, the pistillate flower is not completely formed and ovules are just starting to develop. Pollen reaching the dry stigmatic surface adheres to the receptive cells, germinates and penetrates the epidermis in aproximately 24 h, and grows through the intercellular spaces of a solid transmitting tissue. In cross-pollination, a sequential arrest of pollen tubes was observed along the style, providing preliminary evidence for a pollen tube competition mechanism. As a consequence, few pollen tubes reach the basal portion of the style. Furthermore, pollen tube growth is a discontinuous process since tubes are arrested in the basal portion of the style about 10–12 days after pollination. While tubes are latent, the ovarian loculus starts to develop from an emerging mass of sporogeneous cells which later will differentiate into the placenta and ovules. One and a half months after pollination ovules complete their differentiation, tubes resume growth and fertilisation occurs. Ovular abortion is frequent at this stage, and only one ovule will successfully mature during autumn into a monospermic seed.

Current Biology, 2003

embryo sacs contain tangled structures ( ) not observed in wild-type plants ( ).

Plant Physiology, 2003

Pollen tubes are a good model for the study of cell growth and morphogenesis because of their ext... more Pollen tubes are a good model for the study of cell growth and morphogenesis because of their extreme elongation without cell division. Yet, knowledge about the genetic basis of pollen germination and tube growth is still lagging behind advances in pollen physiology and biochemistry. In an effort to reduce this gap, we have developed a new method to obtain highly purified, hydrated pollen grains of Arabidopsis through flowcytometric sorting, and we used GeneChips (Affymetrix, Santa Clara, CA; representing approximately 8,200 genes) to compare the transcriptional profile of sorted pollen with those of four vegetative tissues (seedlings, leaves, roots, and siliques). We present a new graphical tool allowing genomic scale visualization of the unique transcriptional profile of pollen. The 1,584 genes expressed in pollen showed a 90% overlap with genes expressed in these vegetative tissues, whereas one-third of the genes constitutively expressed in the vegetative tissues were not expressed in pollen. Among the 469 genes enriched in pollen, 162 were selectively expressed, and most of these had not been associated previously with pollen. Their functional classification reveals several new candidate genes, mainly in the categories of signal transduction and cell wall biosynthesis and regulation. Thus, the results presented improve our knowledge of the molecular mechanisms underlying pollen germination and tube growth and provide new directions for deciphering their genetic basis. Because pollen expresses about one-third of the number of genes expressed on average in other organs, it may constitute an ideal system to study fundamental mechanisms of cell biology and, by omission, of cell division. ; fax 351-21-4407970.

Journal of Experimental Botany, 2010

The process of pollen germination and tube growth in the pistil involves a series of cell-cell in... more The process of pollen germination and tube growth in the pistil involves a series of cell-cell interactions, some facilitating fertilization while others prohibiting pollen tube access to the female gametophyte, either because of incompatibility or as a result of mechanisms to avert polyspermy and to ensure reproductive success. Understanding pollen tube growth and guidance to the female gametophyte has long been a pursuit among plant biologists, and observations indicate that diverse strategies may be adopted by different plant species. Recent studies in Arabidopsis, maize, and Torenia fournieri suggest that low molecular weight secretory molecules probably play major roles in the short-range attraction of pollen tubes to the female gametophyte. The process of pollen tube growth in the pistil occurs beneath several cell layers so much of the information that conveys the intimate partnership between penetrating pollen tubes and the female tissues has come from fixed samples and observations of in vitro pollen tube growth responses to female factors. A unique glimpse of the in vivo pollen germination and tube growth process is provided here by intra-vital two-photon excitation (TPE) microscopy of pollinated Arabidopsis pistils that remained on intact plants. Further discoveries of critical factors of male or female origins and how they control the pollen tube growth and fertilization process will broaden our understanding of the common themes and diverse strategies that plants have evolved to ensure reproductive success. The advancement of imaging technology to monitor pollination and fertilization and the development of probes to monitor various aspects of the pollen tube growth process, including pollen intracellular dynamics, will allow us to superimpose details obtained from studying pollen tube growth in culture conditions to interpret and understand the in vivo events.

Sexual Plant Reproduction, 2001

Intraspecific barriers promote outcrossing while interspecific mechanisms may contribute to the i... more Intraspecific barriers promote outcrossing while interspecific mechanisms may contribute to the isolation of species; both control the exchange of genes between plants. In this paper we establish that post-pollination mechanisms promote outcross and act at different temporal and spatial levels to control seed set and quality in Quercus species. Controlled pollinations were performed to investigate intraspecific crossing barriers in Q. suber and pollen-pistil interactions following interspecific pollinations with some simpatric Quercus species. Cytological data of intraspecific crosses in Q. suber and Q. ilex have shown different kinetics on pollen tube growth after self and outcross pollination, but pollen tubes were able to reach the base of the styles in both species and seed set was successful. Although some pre-zygotic interaction is occurring at the style, the most important interaction takes place at ovary. The cross between Q. ilex and Q. suber is possible only in one direction, revealing a case of unilateral incongruity. We show that the lack of seed set observed in the cross Q. suber×Q. ilex is due to the inability of pollen tubes to penetrate the transmitting tissue after germination. With Q. suber mainly as female parent, pollinations with other simpatric Quercus species have shown different levels of constraint on pollen tube progression at stigma and style levels. Eventual hybridisation between Quercus species will depend on the compatibility of pollen-pistil interactions, on the competitive ability of pollen genotypes, and, most important, on the overlapping of geographical, phenological and ecological factors. Differences in seed set and seed allocation was evident in all crosses, and was particularly outstanding in interspecific and in self intraspecific crosses, determining the ultimate level of seed production and quality in Quercus species.

Plant Cell, 2008

Pollen tubes must navigate through different female tissues to deliver sperm to the embryo sac fo... more Pollen tubes must navigate through different female tissues to deliver sperm to the embryo sac for fertilization. Protein disulfide isomerases play important roles in the maturation of secreted or plasma membrane proteins. Here, we show that certain T-DNA insertions in Arabidopsis thaliana PDIL2-1, a protein disulfide isomerase (PDI), have reduced seed set, due to delays in embryo sac maturation. Reciprocal crosses indicate that these mutations acted sporophytically, and aniline blue staining and scanning electron microscopy showed that funicular and micropylar pollen tube guidance were disrupted. A PDIL2-1-yellow fluorescent protein fusion was mainly localized in the endoplasmic reticulum and was expressed in all tissues examined. In ovules, expression in integument tissues was much higher in the micropylar region in later developmental stages, but there was no expression in embryo sacs. We show that reduced seed set occurred when another copy of full-length PDIL2-1 or when enzymatically active truncated versions were expressed, but not when an enzymatically inactive version was expressed, indicating that these T-DNA insertion lines are gain-of-function mutants. Our results suggest that these truncated versions of PDIL2-1 function in sporophytic tissues to affect ovule structure and impede embryo sac development, thereby disrupting pollen tube guidance.

Plant Journal, 2007

Despite much effort, a robust protocol for in vitro germination of Arabidopsis thaliana pollen ha... more Despite much effort, a robust protocol for in vitro germination of Arabidopsis thaliana pollen has been elusive. Here we show that controlled temperatures, a largely disregarded factor in previous studies, and a simple optimized medium, solidified or liquid, yielded pollen germination rates above 80% and pollen tube lengths of hundreds of microns, with both Columbia and Landsberg erecta (Ler) ecotypes. We found that pollen germination and tube growth were dependent on pollen density in both liquid and solid medium. Pollen germination rates were not substantially affected by flower or plant age. The quartet1 mutation negatively affected pollen germination, especially in the Ler ecotype. This protocol will facilitate functional analyses of insertional mutants affecting male gametophyte function, and should allow detailed gene expression analyses during pollen tube growth. Arabidopsis thaliana can now be included on the list of plant species that are suitable models for physiological studies of pollen tube elongation and tip growth.

Genetics, 2009

Functional analyses of the Arabidopsis genome require analysis of the gametophytic generation, si... more Functional analyses of the Arabidopsis genome require analysis of the gametophytic generation, since about 10% of the genes are expressed in the male gametophyte and about 9% in the female gametophyte. Here we describe the genetic and molecular characterization of 67 Ds insertion lines that show reduced transmission through the male gametophyte. About half of these mutations are male gametophytic-specific mutations, while the others also affect female transmission. Genomic sequences flanking both sides of the Ds element were recovered for 39 lines; for 16 the Ds elements were inserted in or close to coding regions, while 7 were located in intergenic/unannotated regions of the genome. For the remaining 16 lines, chromosomal rearrangements such as translocations or deletions, ranging between 30 kb to 500 kb, were associated with the transposition event. The mutants were classified into five groups according to the developmental processes affected; these ranged from defects in early stages of gametogenesis to later defects affecting pollen germination, pollen tube growth, polarity or guidance, or pollen tube-embryo sac interactions or fertilization. The isolated mutants carry Ds insertions in genes with diverse biological functions and potentially specify new functions for several unannotated or unknown proteins.

International Journal of Developmental Biology, 2005

The evolutionary success of higher plants relies on a very short gametophytic phase, which underl... more The evolutionary success of higher plants relies on a very short gametophytic phase, which underlies the sexual reproduction cycle. Sexual plant reproduction takes place in special organs of the flower: pollen, the male gametophyte, is released from the anthers and then adheres, grows and interacts along various tissues of the female organs, collectively known as the pistil. Finally, it fertilizes the female gametophyte, the embryo sac. Pollen is released as bi or tricellular, highly de-hydrated and presumably containing all the biochemical components and transcripts to germinate. Upon hydration on the female tissues, it develops a cytoplasmic extension, the pollen tube, which is one of the fastest growing cells in nature. Pollen is completely "ready-to-go", but despite this seemingly simple reaction, very complex interactions take place with the female tissues. In higher animals, genetic mechanisms for sex determination establish striking developmental differences between males and females. In contrast, most higher plant species develop both male and female structures within the same flower, allowing self-fertilization. Outcrossing is ensured by self-incompatibility mechanisms, which evolved under precise genetic control, controlling self-recognition and cell-to-cell interaction. Equally important is pollen selection along the female tissues, where interactions between different cell types with inherent signalling properties correspond to check-points to ensure fertilization. Last but not least, pollen-pistil interaction occurs in a way that enables the correct targeting of the pollen tubes to the receptive ovules. In this review, we cover the basic mechanisms underlying sexual plant reproduction, from the structural and cellular determinants, to the most recent genetic advances.

International Journal of Developmental Biology, 2005

Higher plants have evolved to be one of the predominant life forms on this planet. A great deal o... more Higher plants have evolved to be one of the predominant life forms on this planet. A great deal of this evolutionary success relies in a very short gametophytic phase which underlies the sexual reproduction cycle. Sexual plant reproduction takes place in special organs of the flower. In most species the processes of gametogenesis, pollination, syngamy and embryogenesis are sequentially coordinated to give rise to a functional seed in a matter of few weeks. Any of these processes is so intricately complex and precisely regulated that it becomes no wonder that each involves more specific genes and cellular processes than any other function in the plant life cycle. While variability generation -the evolutionary output of the sexual cycle -is the same as in any other Kingdom, plants do it using a completely original set of mechanisms, many of which are not yet comprehended. In this paper, we cover the fundamental features of male and female gametogenesis. While the physiological and cellular bases of these processes have been continuously described since the early nineteen century, recent usage of Arabidopsis and other species as central models has brought about a great deal of specific information regarding their genetic regulation. Transcriptomics has recently enlarged the repertoire and pollen became the first gametophyte to have a fully described transcriptome in plants. We thus place special emphasis on the way this newly accumulated genetic and transcriptional information impacts our current understanding of the mechanisms of gametogenesis.

Sexual Plant Reproduction, 1999

Cork oak (Quercus suber L.) is a monoecious wind-pollinated species with a protandrous system to... more Cork oak (Quercus suber L.) is a monoecious wind-pollinated species with a protandrous system to ensure cross-pollination. To the best of our knowledge, this report provides the first insight into the sexual reproduction cycle in this species. The cork oak flowering season extends from April until the end of May. Our results show that, at anthesis, the pistillate flower is not completely formed and ovules are just starting to develop. Pollen reaching the dry stigmatic surface adheres to the receptive cells, germinates and penetrates the epidermis in aproximately 24 h, and grows through the intercellular spaces of a solid transmitting tissue. In cross-pollination, a sequential arrest of pollen tubes was observed along the style, providing preliminary evidence for a pollen tube competition mechanism. As a consequence, few pollen tubes reach the basal portion of the style. Furthermore, pollen tube growth is a discontinuous process since tubes are arrested in the basal portion of the style about 10–12 days after pollination. While tubes are latent, the ovarian loculus starts to develop from an emerging mass of sporogeneous cells which later will differentiate into the placenta and ovules. One and a half months after pollination ovules complete their differentiation, tubes resume growth and fertilisation occurs. Ovular abortion is frequent at this stage, and only one ovule will successfully mature during autumn into a monospermic seed.

Current Biology, 2003

embryo sacs contain tangled structures ( ) not observed in wild-type plants ( ).

Plant Physiology, 2003

Pollen tubes are a good model for the study of cell growth and morphogenesis because of their ext... more Pollen tubes are a good model for the study of cell growth and morphogenesis because of their extreme elongation without cell division. Yet, knowledge about the genetic basis of pollen germination and tube growth is still lagging behind advances in pollen physiology and biochemistry. In an effort to reduce this gap, we have developed a new method to obtain highly purified, hydrated pollen grains of Arabidopsis through flowcytometric sorting, and we used GeneChips (Affymetrix, Santa Clara, CA; representing approximately 8,200 genes) to compare the transcriptional profile of sorted pollen with those of four vegetative tissues (seedlings, leaves, roots, and siliques). We present a new graphical tool allowing genomic scale visualization of the unique transcriptional profile of pollen. The 1,584 genes expressed in pollen showed a 90% overlap with genes expressed in these vegetative tissues, whereas one-third of the genes constitutively expressed in the vegetative tissues were not expressed in pollen. Among the 469 genes enriched in pollen, 162 were selectively expressed, and most of these had not been associated previously with pollen. Their functional classification reveals several new candidate genes, mainly in the categories of signal transduction and cell wall biosynthesis and regulation. Thus, the results presented improve our knowledge of the molecular mechanisms underlying pollen germination and tube growth and provide new directions for deciphering their genetic basis. Because pollen expresses about one-third of the number of genes expressed on average in other organs, it may constitute an ideal system to study fundamental mechanisms of cell biology and, by omission, of cell division. ; fax 351-21-4407970.