All organisms have been subjected to and have evolved with the ubiquitous force of gravity, and most exhibit the ability to sense and respond to this stimulus. To simplify an investigation of the molecular components of a cell's gravity response, this dissertation employs the single-celled spores of the fern Ceratopteris richardii. These spores have a polar calcium flux that is determined by the gravity vector, but an understanding of what the molecular components driving this flux are and how they influence subsequent developmental processes is lacking. Of the possible molecular components, available literature pointed to Ca²⁺-ATPase transporters as an obvious key participant and so they were selected as the main molecule of investigation. Our results describe the first cloned Ca²⁺-ATPase from C. richardii, CrACA1. CrACA1 has high similarity to known plant Ca²⁺-ATPases, specifically plasma membrane (PM) Ca²⁺-ATPases from Arabidopsis, and exhibits in vivo Ca²⁺-ATPase activity. An improved method for the statistical analysis and presentation of qualitative RT-PCR data was employed. The RNA, as well as the protein, of CrACA1 is present during the polarity fixation window which supported the need for further analyses of the role of Ca²⁺-ATPases. Our results showing that Ca²⁺-ATPase inhibitors significantly alter the gravity-directed calcium flux of spores are consistent with previous work but offer valuable new insights. The spore PM Ca²⁺-ATPases have large impacts on the calcium flux and rhizoid growth but no appreciable impact on polar axis alignment. The results on endomembrane-type Ca²⁺-ATPases make it clear that this class of pumps has major roles in both axis alignment and tip growth; rhizoid growth is inhibited but alignment to the gravity vector is improved. The updated model for gravity perception responses in C. richardii spores places a strong emphasis on calcium channels and Ca²⁺-ATPases working in concert to result in a bottom-localized calcium pool to align the polar axis with hints of store-operated calcium mobilization. The work presented represents an increase in our knowledge of one way a single cell can respond to the force of gravity, offering testable hypotheses to further refine gravity perception models incorporating calcium localization. / text

Thesis (Ph.D.)--Ohio University, November, 2007. / Title from PDF t.p. Includes bibliographical references. Also available online via OhioLINK's ETD Center: http://www.ohiolink.edu/etd/view.cgi?ohiou1188579264

The life cycle of all land plants is characterized by the alternation between two multicellular generations: haploid gametophytes and diploid sporophytes. Whereas meiotic recombination and fertilization via sexual reproduction is known to confer adaptive evolutionary benefits, some plant species can reproduce asexually bypassing meiosis and fertilization yet still undergo alternation of generations. In ferns, the two asexual reproductive pathways are termed apogamy and apospory, i.e. the asexual reproduction of sporophytes and gametophytes, respectively. Although understanding the process of asexual reproduction in flowering plants would be of considerable agricultural value, the molecular mechanisms underlying this process have yet to be identified. Similarly, the genes in ferns that underlie apogamy and apospory are unknown. Knowledge of such genes will provide invaluable information not only for the evolutionary study of asexual reproduction in land plants, but also insight into the developmental program for each generation. My PhD research focuses on the investigation of candidate genes involved in asexual reproduction in the model fern Ceratopteris richardii. Though the molecular mechanisms underlying asexual reproduction are not understood, it is hypothesized that asexual reproduction is the result of altered regulation of certain sexual reproduction-related genes. Therefore, I selected candidate genes for asexual reproduction in C. richardii, CrEMS1, CrKNOX3 and CrANT, based on the functions of their respective homologs during sexual reproduction in two other model systems: Arabidopsis thaliana and Physcomitrella patens. I showed that, in C. richardii, CrEMS1 is expressed during key events of sexual reproduction, including sporogenesis, gametogenesis and embryo development; CrKNOX3 expression is specific for the mature egg cells and CrANT expression is restricted to sperm cells. The presence of CrEMS1 during early embryo development made it a suitable candidate for an embryonic marker for future research. To study the function of the candidate genes, I overexpressed CrKNOX3 and BnBBM, a flowering plant BBM gene that has similar expression patterns in Brassica to CrANT. The overexpression of CrKNOX3 results in unique cell outgrowth from the antheridia, and overexpression of BnBBM results in spontaneous apogamy induction. Whole mount in situ also showed CrEMS1 expression, marking embryonic identity, in pre-apogamous cells undergoing sugar-induced and overexpression of BnBBM-induced apogamy. The results of my PhD research provides, for the first time, insight into the molecular mechanisms underlying asexual reproduction in the model fern C. richardii, and invaluable tools, transient and stable Agrobacterium-mediated transformation protocols, for functional genomics research in this fern. The spontaneous apogamy induction on BnBBM-expressing gametophytes and the presence of CrEMS1 in pre-apogamous cells confirm the hypothesis that asexual reproduction is the result of the alteration in expression of sexual-related genes, verifying that the two pathways share a common set of genes.

O aerênquima é um tecido vegetal com grandes lacunas de ar que podem ter função de circulação de gases e/ou sustentação de órgãos, estando presente nas raízes e órgãos aéreos de plantas aquáticas ou sob alagamento, podendo ainda ser formado em decorrência de algum estresse. Ceratopteris richardii é uma planta aquática, de hábito anfíbio que apresenta aerênquima em seus órgãos vegetativos. Dessa forma, esse trabalho buscou analisar a formação de aerênquima nas raízes e pecíolo desta espécie frente a uma situação de restrição hídrica e verificar sua plasticidade fenotípica. Vinte indivíduos de C. richardii foram cultivados e submetidos a um tratamento com diferentes concentrações de PEG 6000. Em todas as amostras analisadas, foi observada a formação de aerênquima. A análise qualitativa mostrou diferença entre os tratamentos, porém, a análise estatística revelou que as diferenças não são significativas para o número amostral empregado. Variações de crescimento do esporófito revelaram que a espécie de estudo modificou algumas características de desenvolvimento em resposta a restrição hídrica. Tais resultados revelam importantes características para esta espécie, que parece aclimatar-se a situação de estresse a qual foi submetida. Com base nisso, sugere-se ampliar estes estudos para elucidar questões relacionadas a formação de aerênquima e o ambiente, seja para esta espécie ou de outro grupo taxonômico. / Aerenchyma is a plant tissue with large air gaps that may have gas circulation function and / or sustain organs. This structure is present in roots and aerial organs of water plants and in plants under flooding it may also be formed during some stress. Ceratopteris richardii is an aquatic plant, with amphibious habit that has aerenchyma in their vegetative organs. Thus, the aim of this study is analyze the aerenchyma formation in roots and stem of this specie under water restriction situation and verify their phenotypic plasticity. Twenty specimen of C. richardii were cultured and subjected to treatment with different concentrations of PEG 6000. In all samples, were observed aerenchyma formation. Qualitative analysis showed differences among treatments, however, the statistical analysis revealed that the differences are not significant for the sample size. Sporophyte growth variations revealed that the specie of study modified some growth characteristics in response to water restriction. These results reveal important characteristics for C. richardii, which seems to acclimatize the stress situation which has submitted. On the base that, it is suggested further studies to become clearer the relation between aerenchyma formation and environment, even to either this species or another taxonomic group.

O aerênquima é um tecido vegetal com grandes lacunas de ar que podem ter função de circulação de gases e/ou sustentação de órgãos, estando presente nas raízes e órgãos aéreos de plantas aquáticas ou sob alagamento, podendo ainda ser formado em decorrência de algum estresse. Ceratopteris richardii é uma planta aquática, de hábito anfíbio que apresenta aerênquima em seus órgãos vegetativos. Dessa forma, esse trabalho buscou analisar a formação de aerênquima nas raízes e pecíolo desta espécie frente a uma situação de restrição hídrica e verificar sua plasticidade fenotípica. Vinte indivíduos de C. richardii foram cultivados e submetidos a um tratamento com diferentes concentrações de PEG 6000. Em todas as amostras analisadas, foi observada a formação de aerênquima. A análise qualitativa mostrou diferença entre os tratamentos, porém, a análise estatística revelou que as diferenças não são significativas para o número amostral empregado. Variações de crescimento do esporófito revelaram que a espécie de estudo modificou algumas características de desenvolvimento em resposta a restrição hídrica. Tais resultados revelam importantes características para esta espécie, que parece aclimatar-se a situação de estresse a qual foi submetida. Com base nisso, sugere-se ampliar estes estudos para elucidar questões relacionadas a formação de aerênquima e o ambiente, seja para esta espécie ou de outro grupo taxonômico. / Aerenchyma is a plant tissue with large air gaps that may have gas circulation function and / or sustain organs. This structure is present in roots and aerial organs of water plants and in plants under flooding it may also be formed during some stress. Ceratopteris richardii is an aquatic plant, with amphibious habit that has aerenchyma in their vegetative organs. Thus, the aim of this study is analyze the aerenchyma formation in roots and stem of this specie under water restriction situation and verify their phenotypic plasticity. Twenty specimen of C. richardii were cultured and subjected to treatment with different concentrations of PEG 6000. In all samples, were observed aerenchyma formation. Qualitative analysis showed differences among treatments, however, the statistical analysis revealed that the differences are not significant for the sample size. Sporophyte growth variations revealed that the specie of study modified some growth characteristics in response to water restriction. These results reveal important characteristics for C. richardii, which seems to acclimatize the stress situation which has submitted. On the base that, it is suggested further studies to become clearer the relation between aerenchyma formation and environment, even to either this species or another taxonomic group.

O aerênquima é um tecido vegetal com grandes lacunas de ar que podem ter função de circulação de gases e/ou sustentação de órgãos, estando presente nas raízes e órgãos aéreos de plantas aquáticas ou sob alagamento, podendo ainda ser formado em decorrência de algum estresse. Ceratopteris richardii é uma planta aquática, de hábito anfíbio que apresenta aerênquima em seus órgãos vegetativos. Dessa forma, esse trabalho buscou analisar a formação de aerênquima nas raízes e pecíolo desta espécie frente a uma situação de restrição hídrica e verificar sua plasticidade fenotípica. Vinte indivíduos de C. richardii foram cultivados e submetidos a um tratamento com diferentes concentrações de PEG 6000. Em todas as amostras analisadas, foi observada a formação de aerênquima. A análise qualitativa mostrou diferença entre os tratamentos, porém, a análise estatística revelou que as diferenças não são significativas para o número amostral empregado. Variações de crescimento do esporófito revelaram que a espécie de estudo modificou algumas características de desenvolvimento em resposta a restrição hídrica. Tais resultados revelam importantes características para esta espécie, que parece aclimatar-se a situação de estresse a qual foi submetida. Com base nisso, sugere-se ampliar estes estudos para elucidar questões relacionadas a formação de aerênquima e o ambiente, seja para esta espécie ou de outro grupo taxonômico. / Aerenchyma is a plant tissue with large air gaps that may have gas circulation function and / or sustain organs. This structure is present in roots and aerial organs of water plants and in plants under flooding it may also be formed during some stress. Ceratopteris richardii is an aquatic plant, with amphibious habit that has aerenchyma in their vegetative organs. Thus, the aim of this study is analyze the aerenchyma formation in roots and stem of this specie under water restriction situation and verify their phenotypic plasticity. Twenty specimen of C. richardii were cultured and subjected to treatment with different concentrations of PEG 6000. In all samples, were observed aerenchyma formation. Qualitative analysis showed differences among treatments, however, the statistical analysis revealed that the differences are not significant for the sample size. Sporophyte growth variations revealed that the specie of study modified some growth characteristics in response to water restriction. These results reveal important characteristics for C. richardii, which seems to acclimatize the stress situation which has submitted. On the base that, it is suggested further studies to become clearer the relation between aerenchyma formation and environment, even to either this species or another taxonomic group.

No description available.

[ES] El correcto desarrollo de tejidos vasculares depende del ajuste preciso entre la proliferación de células vasculares y la diferenciación celular. En Arabidopsis thaliana, la proliferación de células vasculares en el cambium es potenciada por la citoquinina, la síntesi de la cual está promovida por la actividad dependiente de auxina de un heterodímero de factores de transcripción (TF) formado por LONESOME HIGHWAY (LHW) y por TARGET OF MONOPTEROS 5 (TMO5). Como mecanismo de seguridad, las auxinas también activan un módulo inhibidor que implica la inducción precisa de la Termospermina (Tspm) sintasa ACAULIS5 (ACL5) en células vasculares proliferantes por acción conjunta de las auxinas y del TF Class III HD-ZIP (C3HDZ) AtHB8. Entonces, la Tspm permite la traducción de las proteínas SACL de forma celular autónoma, que perjudican la actividad de LHW. Sin embargo, la observación de que estos elementos están presentes en los genomas de todas las plantas terrestres - y no sólo de las plantas vasculares - plantea dos preguntas desde una perspectiva evolutiva: (i) ¿cuál es la función de estos genes en las plantas terrestres no vasculares? y (ii) ¿cuándo se creó el módulo regulador concreto? En esta Tesis, mediante la combinación de análisis filogenéticos, celulares y moleculares con la hepática Marchantia polymorpha, proponemos que la auxina y C3HDZ son reguladores ancestrales de la expresión de ACL5, y que esta conexión se mantiene en las traqueófitas y las briófitas existentes. Por el contrario, la traducción dependiente de Tspm de SACL parece ser específica de las traqueófitas, basado en la aparición de un uORF conservado en la secuencia 5' líder de los tránscritos de SACL y en evidencia experimental basada en ensayos transitorios para la traducción de SACL. De acuerdo con estas observaciones, las funciones de MpACL5 y MpSACL son diferentes en M. polymorpha. MpACL5 se expresa en "notches" apicales y modula la bifurcación de los meristemos. Por otro lado, la expresión de MpSACL está mayoritariamente excluida de los "notches" apicales y su actividad afecta negativamente la producción de gemas y rizoides mediante la interacción con MpRSL1. Finalmente, la hibridación de ARN in situ de ortólogos de C3HDZ, ACL5 y SACL en la gimnosperma Ginkgo biloba, el helecho Ceratopteris richardii y la licófita Selaginella kraussiana indican que la expresión de los tres genes se solapa en los tejidos vasculares. Nuestros resultados sugieren que la función de C3HDZ, ACL5 y SACL ha seguido trayectorias evolutivas divergentes en briófitas y traqueófitas, para controlar, finalmente, diferentes funciones específicas dentro de cada linaje. Sólo en las traqueófitas se formó el módulo regulador y se asoció con la restricción de la proliferación de células vasculares. / [CA] El correcte desenvolupament dels teixits vasculars depèn del precís ajust entre la proliferació de cèl·lules vasculars i la diferenciació cel·lular. En Arabidopsis thaliana, la proliferació de cèl·lules vasculars al càmbium és potenciada per la citoquinina, la síntesi de la qual està promoguda per l'activitat dependent d'auxina d'un heterodímer de factors de transcripció (TF) format per LONESOME HIGHWAY (LHW) i TARGET OF MONOPTEROS 5 (TMO5). Com a mecanisme de seguretat, l'auxina també activa un mòdul inhibidor que implica la inducció precisa de la Termospermina (Tspm) sintasa ACAULIS5 (ACL5) en cèl·lules vasculars proliferants per l'acció conjunta de l'auxina i del TF Class III HD-ZIP (C3HDZ) AtHB8. Llavors, la Tspm permet la traducció de les proteïnes SACL de forma autònoma cel·lular, que perjudiquen l'activitat de LHW. Tanmateix, l'observació de que aquests elements estan presents en els genomes de totes les plantes terrestres - i no només de les plantes vasculars - planteja dues preguntes des d'una perspectiva evolutiva: (i) quina és la funció d'aquests gens en les plantes terrestres no vasculars? i (ii) quan es va crear el mòdul regulador complet? En aquesta Tesi, mitjançant la combinació d'anàlisis filogenètics, cel·lulars i moleculars amb la hepàtica Marchantia polymorpha, proposem que l'auxina i C3HDZ són reguladors ancestrals de l'expressió d'ACL5, i que aquesta connexió es mantén en els traqueòfits i briòfits existents. Per contra, la traducció depenent de Tspm de SACL sembla ser específica dels traqueòfits, basat en l'aparició d'un uORF conservat a la seqüència 5' líder dels trànscrits de SACL i en evidència experimental basada en assajos transitoris per a la traducció de SACL. D'acord amb aquestes observacions, les funcions de MpACL5 i MpSACL són diferents a M. polymorpha. MpACL5 s'expressa en "notch" apicals i modula la bifurcació dels meristems. D'altra banda, l'expressió de MpSACL està majoritàriament exclosa dels "notch" apicals i la seva activitat afecta negativament la producció de gemmes i rizoids mitjançant la interacció amb MpRSL1. Finalment, la hibridació d'ARN in situ d'ortòlegs de C3HDZ, ACL5 i SACL a la gimnosperma Ginkgo biloba, la falguera Ceratopteris richardii i el licòfit Selaginella kraussiana indica que l'expressió dels tres gens es solapa als teixits vasculars. Els nostres resultats suggereixen que la funció de C3HDZ, ACL5 i SACL va seguir trajectòries evolutives divergents en briòfits i traqueòfits, per controlar, finalment, diferents funcions específiques dins de cada llinatge. Només en els traqueòfits es va formar el mòdul regulador i es va associar amb la restricció de la proliferació de cèl·lules vasculars. / [EN] The correct development of vascular tissues depends on the precise adjustment between vascular cell proliferation and cell differentiation. In Arabidopsis thaliana, vascular cell proliferation in the cambium is enhanced by cytokinin, whose synthesis is promoted by the auxin-dependent activity of a transcription factor (TF) heterodimer formed by LONESOME HIGHWAY (LHW) and TARGET OF MONOPTEROS 5 (TMO5). As a safety mechanism, auxin also deploys a negative feedforward regulatory module which involves the precise induction of the Thermospermine (Tspm) synthase ACAULIS5 (ACL5) in proliferating vascular cells by the joint action of auxin and the class-III HD-ZIP (C3HDZ) AtHB8 TF. Tspm then allows the cell-autonomous translation of the SACL proteins, which impair the activity of LHW. However, the observation that these elements are present in the genomes of all land plants -and not only vascular plants- poses two questions from an evolutionary perspective: (i) what is the function of these genes in non-vascular land plants? and (ii) when was the full regulatory module assembled? In this Thesis, through the combination of phylogenetic, cellular, and molecular genetic analyses with the liverwort Marchantia polymorpha, we propose that auxin and C3HDZ are ancestral regulators of ACL5 expression, and that this connection is maintained in extant tracheophytes and bryophytes. On the contrary, thermospermine-dependent translation of SACL seems to be specific of tracheophytes, based on the appearance of a conserved uORF in the 5' leader sequence of SACL transcripts and on experimental evidence using transient assays for SACL translation. In agreement with these observations, the functions of MpACL5 and MpSACL are different in M. polymorpha. MpACL5 is expressed in apical notches and modulates meristem bifurcation. On the other hand, MpSACL expression is mostly excluded from apical notches and its activity negatively affects gemmae and rhizoid production through the interaction with MpRSL1. Finally, in situ RNA hibridization of C3HDZ, ACL5 and SACL orthologs in the gymnosperm Ginkgo biloba, the fern Ceratopteris richardi and the lycophyte Selaginella kraussiana indicates that the expression of the three genes overlaps in vascular tissues. Our results suggest that the function of C3HDZ, ACL5 and SACL followed divergent evolutionary trajectories in bryophytes and tracheophytes, to ultimately control different lineage-specific functions. Only in tracheophytes was the regulatory module assembled and associated with the restriction of vascular cell proliferation. / Solé Gil, A. (2023). Origin & Evolution of the C3HDZ-ACL5-SACL Regulatory Module in Land Plants [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/196681