Somatic embryogenesis can provide researchers with an important tool to study the physiological and molecular mechanisms involved in embryo development. In spruce, few lines are able to produce fully developed embryos due to the presence of malformed meristems. Genes from two families known as KNOX (knotted-like homeobox) and ARGONAUTE (AGO) have previously been found to be involved in meristem development and maintenance. This work documents the discovery of a new member of the AGO family of proteins designated as PgAGO and the further study of a KNOX gene known as HBK2. The complete coding sequence of both PgAGO and HBK2 was obtained through screening of cDNA libraries generated from white spruce (Picea glauca) somatic embryos. RNA in-situ hybridization studies showed that PgAGO mRNAs accumulate preferentially within cells of the shoot and root apical meristems in developing spruce embryos. In addition, the expression of PgAGO was low in white spruce lines unable to produce embryos in culture. Norway spruce (Picea abies) embryogenic tissue was transformed via microprojectile bombardment with an antisense construct of PgAGO. Down-regulation of PgAGO altered proper development of the apical meristems and reduced embryo regeneration. RNA in-situ hybridization studies showed that HBK2 is specifically expressed in the sub-apical and cortical regions of developing embryos. Like PgAGO, HBK2 expression was diminished in white spruce lines unable to produce embryos in culture. Transformation experiments with antisense constructs of HBK2 completely arrested somatic embryo development. This study reveals the importance of a functional meristem during embryo development.

Somatic embryogenesis can provide researchers with an important tool to study the physiological and molecular mechanisms involved in embryo development. In spruce, few lines are able to produce fully developed embryos due to the presence of malformed meristems. Genes from two families known as KNOX (knotted-like homeobox) and ARGONAUTE (AGO) have previously been found to be involved in meristem development and maintenance. This work documents the discovery of a new member of the AGO family of proteins designated as PgAGO and the further study of a KNOX gene known as HBK2. The complete coding sequence of both PgAGO and HBK2 was obtained through screening of cDNA libraries generated from white spruce (Picea glauca) somatic embryos. RNA in-situ hybridization studies showed that PgAGO mRNAs accumulate preferentially within cells of the shoot and root apical meristems in developing spruce embryos. In addition, the expression of PgAGO was low in white spruce lines unable to produce embryos in culture. Norway spruce (Picea abies) embryogenic tissue was transformed via microprojectile bombardment with an antisense construct of PgAGO. Down-regulation of PgAGO altered proper development of the apical meristems and reduced embryo regeneration. RNA in-situ hybridization studies showed that HBK2 is specifically expressed in the sub-apical and cortical regions of developing embryos. Like PgAGO, HBK2 expression was diminished in white spruce lines unable to produce embryos in culture. Transformation experiments with antisense constructs of HBK2 completely arrested somatic embryo development. This study reveals the importance of a functional meristem during embryo development.

Zugl.: Halle (Saale), Univ., Diss., 2008

Universiẗat, Diss., 1999--Köln.

Plant development is an iterative process of organ formation from the primary meristems of the plant. Meristem activity is driven by dynamic transcriptional programs that determine cell fate and identity as cells are displaced trough the meristematic tissue to initiate organ primordia. This regulatory network includes members of the TCP and KNOX family of transcription factors, and integrates external and intrinsic cues to efficiently adapt meristem activity to an ever-changing environment. However, how this integration occurs is not clear yet. DELLA proteins have been proposed to modulate transcriptional circuits in plants in response to environmental signals. Although they do not show DNA binding capacity, DELLAs regulate transcription through physical interaction with a large number of DNA-binding transcription factors and other transcriptional regulators. Given the observed interaction between DELLAs and several members of the TCP family of transcription factors, we have explored the relevance of this interaction in the regulation of primary meristems. We have confirmed that DELLAs interact with members of both Class I and Class II TCPs, and prevent their ability to regulate downstream targets. In the embryonic roots, DELLAs maintain a dormant meristem by impairing TCP14/15-dependent activation of cell-cycle genes. On the other hand, DELLAs participate in the establishment of the shoot apical meristem domain that keeps an indeterminate fate, through the control of KNAT1 gene expression by the TCP2/4-AS1 regulatory module. In summary, this Thesis provides a mechanistic framework to eventually explain environmental regulation of meristem activity. / El desarrollo de las plantas es un proceso iterativo de formación de órganos a partir de los meristemos primarios de la planta. La actividad meristemática está dirigida por programas transcripcionales dinámicos que determinan el destino y la identidad celular conforme las células son desplazadas a través del tejido meristemático para iniciar el primordio del órgano. Esta red regulatoria incluye miembros de las familias de factores de transcripción TCP y KNOX, e integra señales externas e intrínsecas para adaptar eficientemente la actividad meristemática al medio ambiente, siempre cambiante. Sin embargo, la manera en que esta integración ocurre no se ha desvelado todavía. Se ha propuesto que en plantas, las proteínas DELLA modulan los circuitos transcripcionales en respuesta a señales medioambientales. Aunque no muestran capacidad de unión al ADN, las DELLAs regulan la transcripción a través de su interacción física con un gran número de factores de transcripción capaces de unirse al ADN y otros reguladores transcripcionales. Dada la interacción observada entre las DELLA y varios miembros de la familia de factores de transcripción TCP, hemos explorado la relevancia de esta interacción en la regulación de los meristemos primarios. Hemos confirmado que las DELLA interaccionan con miembros de las dos clases de TCPs (Clase I y Clase II) e impiden su capacidad de regular dianas aguas abajo. En la raíz del embrión, las DELLAs mantienen el meristemo durmiente al impedir la activación de los genes de ciclo celular dependiente del módulo TCP14/15. Por otro lado, las DELLAs participan en el establecimiento del meristemo apical del tallo, que mantiene un estado indiferenciado, a través del control el módulo TCP2/4-AS1, el cual regula la expresión del gen KNAT1. En resumen, esta Tesis aporta un marco mecanístico para explicar, con el tiempo, la regulación medioambiental de la actividad meristemática. / El desenvolupament de les plantes consiteix en un procés iteratiu de formació d'órgans a partir dels meristems primaris. L'activitat meristemàtica està diridida per programes transcripcionals dinàmics que determinen el destí i la identitat cel.lular a mesura que les cèl.lules es van allunyant del meristem per formar els primordis d`órgans. Esta xarxa de regulació inclou membres de les famílies de factors de transcripció TCP i KNOX, i integra senyals externes i intrínseques per adaptar d'una manera eficient l'activitat del meristem als canvis del medi ambient. No obstant, no es coneix de quina manera la planta fa esta integració. S'ha proposat que les proteïnes DELLA modulen estes xarxes transcripcionals en resposta a senyals del medi. Estes proteïnes no tenen capacitat d'unir-se a l'ADN, però regulen la transcripció mitjançant la interacció amb factors de transcripció i altres reguladors transcripcionals. Donada la interacció entre les proteïnes DELLA i alguns membres de la família de factors de transcripció TCP, hem explorat la rellevància d'esta interacció a la regulació dels meristems primaris. Hem confirmat que les DELLA interaccionen amb membres de les dos classes de TCPs (Classe I i Classe II) i els impedeixen regular les seues dianes. A l'arrel de l'embrió, les DELLA mantenen el meristem dorment al impedir l'activació de gens del cicle cel.lular depenent del mòdul TCP14/15. Per una altra banda, les DELLA particípen a l'establiment del meristem apical de la tija, al que mantenen en un estat indiferenciat, mitjançant el control del mòdul TCP2/4-AS1, que regula l'expressió de KNAT1. En resum, esta Tesi aporta un marc mecanístic per poder explicar, més endavant, la regulació mediambiental de l'activitat meristemàtica. / Felipo Benavent, A. (2017). Modulation of primary meristem activity by gibberellins through DELLA-TCP interaction in Arabidopsis [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/82237 / TESIS

Thesis (Ph.D.) - University of Queensland, 2004. / Includes bibliography.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A mamoneira (Ricinus communis) à uma oleaginosa com valor econÃmico considerÃvel devido à composiÃÃo do Ãleo de suas sementes. No entanto, hà algumas limitaÃÃes no seu uso para alimentaÃÃo humana e animal devido à presenÃa de proteÃnas tÃxicas e alergÃnicas, as quais podem ser solucionadas ou minimizadas atravÃs da engenharia genÃtica. Contudo, essa cultura à recalcitrante para tÃcnicas de cultura de tecidos limitando a geraÃÃo de plantas transformadas e os protocolos existentes nÃo sÃo bem estabelecidos. O objetivo desse trabalho foi avaliar a aplicabilidade de um protocolo de regeneraÃÃo existente na literatura à cultivar Nordestina; determinar a via de regeneraÃÃo e avaliar, atravÃs de tÃcnicas histoquÃmicas, as cÃlulas transformadas por biobalÃstica em torno do meristema. Para este propÃsito, eixos embrionÃrios foram cultivados em meio Murashig and Skoog (MS) suplementado com concentraÃÃes crescentes de tidiazuron (TDZ) (0,1; 0,5; 1,0; 2,0; 5,0 e 10,0 mg.L-1). Observou-se que entre as concentraÃÃes de TDZ avaliadas a que apresentou a melhor resposta foi 0,1 mg.L-1, apresentando, em mÃdia, apÃs 56 dias de incubaÃÃo,7,42 partes aÃreas por explante. Ãcido giberÃlico (0,1 mg.L-1) e Ãcido-indol-3 butÃrico (1,0 mg.L-1) foram utilizados na tentativa de alongar e promover o enraizamento, respectivamente, das partes aÃreas obtidas, contudo, nas concentraÃÃes utilizadas, nÃo evidenciou-se nenhum efeito com estes reguladores de crescimento. AnÃlises histolÃgicas revelaram que as partes aÃreas foram formadas de meristemas prÃ-existentes e neo-formados indicando organogÃnese direta. Observaram-se cÃlulas transformadas pelo menos atà a segunda camada celular. AnÃlises da expressÃo estÃvel do gene gus mostraram expressÃo do mesmo, 19 dias apÃs o bombardeamento. Nas anÃlises histolÃgicas foram observadas cÃlulas transformadas em divisÃo. Como conclusÃo a cultivar Nordestina foi capaz de formar mÃltiplas partes aÃreas em meio contendo TDZ e este protocolo de regeneraÃÃo foi compatÃvel com um protocolo de transformaÃÃo por biobalÃstica / Castor bean (Ricinus communis) is an oilseed crop with considerable economic value because of the composition of its oil seed. However, there are some constraints in your use for human and animal food because of the presence of toxic and allergenic proteins. This problem could be solved or reduced through genetic engineering. However, this crop is recalcitrant to tissue culture techniques, thus limiting the formation of transformed plants, and the protocols existing are not well established. The aim of this work was to test the applicability of the regeneration protocol which has been published for the Nordestina cultivar; to determine the regeneration way and to verify trough histochemical techniques, the transformed cells around the meristem. For this purpose, embryo axes were cultured on Murashig and Skoog (MS) medium supplemented with thiadiazuron (TDZ) (0,1; 0,5; 1,0; 2,0; 5,0 e 10,0 mg.L-1). Between the TDZ concentrations tested, 0,1 mg.L-1 presented the better results after 56 days of incubation with 7,42 shoots per explant. Gibberellic acid (GA3) (0,1 mg.L-1) and indole-3-butyric acid (1,0 mg.L-1) were used for elongation and rooting, respectively, of the shoots obtained, however, in the concentrations used, any effect was evidenced. Histological analysis revealed that the shoots were formed by pre existing or neo formed meristems indicating direct organogenesis. Transformed cells were viewed at least in the second cellular layer. Analysis for stable gus expression showed gus expression 19 days after bombardment. In the histological analysis transformed cells in process of division were observed. In conclusion the Nordestina cultivar formed multiple shoots on medium supplemented with TDZ and this protocol of regeneration was compatible with a protocol for biolistic-mediated transformation

Rice have highly derived florets borne on a short branch called ‘spikelet’ comprised of a pair of rudimentary glumes and sterile lemma (empty glumes) that subtends a single fertile floret. The floral organs consist of a pair of lodicules, six stamens and a central carpel that are enclosed by a pair of bract-like organs, called lemma and palea. A progressive reprogramming of meristem identity during the floral development of flowers, on branches on the inflorescence, is correlated with changes in transcriptional status of regulatory genes that execute cascades of distinct developmental events. On the other hand phytohormones such as auxin and cytokinin that are critical in predetermining the sites of new organ primordia emergence and in maintaining the size or populations of meristems. Molecular genetic analyses of mutants have expanded the repository of genes regulating floral organ specification and identity, yet the finer mechanistic details on process downstream to these regulatory genes and co-ordination with phytohormone signalling pathways needs further investigation. One aim of the study presented in this thesis is to develop a tool that would display of spatial description of dynamic auxin or cytokinin accumulation in developing rice inflorescence and floral meristems and to evaluate auxin distribution defects of OsMADS1-RNAi florets using this tool. Additionally, we aim to understand the regulatory effects on OsMADS1 on candidate floral organ and meristem fate determining genes during two temporal phases of flower development to decipher other regulatory cascades controlled by OsMADS1. Spatial distribution profile of phytohormones in young and developing meristems of rice Cytokinin promotes meristem activity (Su et al., 2011) while auxin accumulation, directed by auxin efflux transport PIN proteins predicts sites of new organ initiation (Reinhardt et al., 2003; van Mourik et al., 2012). Previous studies in the lab deciphered that OsMADS1 exerts positive regulatory effects on genes in auxin pathways and repressive effects on cytokinin signaling and biosynthetic genes (Khanday et al., 2013). Thus, the need for a reliable system to understand auxin and cytokinin activity in live inflorescence and floral meristems of rice motivated us to raise promoter: reporter tools to map the spatial and temporal phytohormone distribution. Confocal live imaging conditions in primary roots of IR4DR-GFP and DR5-CyPet lines was performed and responsiveness of the DR5 elements to auxin was authenticated. Auxin maxima were distinctly seen in the epidermal and sub-epidermal cells of inflorescence branch primordia anlagen and apices of newly emerged branch primordia. As floral organs were being initiated, on the floret meristem, we discerned the sequential appearance of auxin accumulation at sites of organ primordia while apices of early floral meristems (FM) showed low auxin content. We clearly detect canalization of auxin streams marking regions of vascular inception. Using this live imaging system we probed auxin patterns and levels in malformed and indeterminate OsMADS1-RNAi florets and we observed a significant reduction in the levels of auxin. Two oppositely positioned peaks of auxin were noted in the persistent FM of OsMADS1-RNAi florets, a pattern similar to auxin dynamics at sites of rudimentary glume primordia on the wild-type (WT) spikelet meristem. These studies were followed up with immunohistochemistry (IHC) on fixed tissues for “PIN” transport proteins that suggest PIN convergence towards organ initiation sites, regions where auxin accumulation was clearly visualized by the IR4DR5-GFP and DR5-CyPet reporters. IHC experiments that detected GFP, in fixed tissues of TCSn-mGFP ER (WT) and TCSn-mGFP ER;OsMADS1-RNAi (OsMADS1-RNAi) inflorescence and florets showed an ectopic increase in the domain of cells with cytokinin response in OsMADS1-RNAi florets, compared to that of WT. Intriguingly, cytokinin responsive cells persisted in the central FM of OsMADS1-RNAi florets that might partially account for some of the FM indeterminacy defects seen in these florets. A correlative observation of these different imaging data hint at some exclusive patterns of the IR4DR5/DR5 and TCSn reporters that in turn lead us to speculate that a cross talk between auxin and cytokinin distribution may contribute to the precise phyllotaxy of lateral organs in rice inflorescence. Studies on novel targets of OsMADS1 in floral organ identity and meristem determinacy Loss of OsMADS1 function results in rice florets with miss specified floral organs and an indeterminate carpel produces new abnormal florets. Despite having several mutants in OsMADS1, mechanisms of how OsMADS1 regulates meristem maintenance and termination is not well understood. Global expression profile in OsMADS1-RNAi vs. WT tissues encompassing a wide range of developing florets (0.2 to 2cm panicles), gave an overview of OsMADS1 functions in many aspects of floret development. Here, a gene-targeted knockout of OsMADS1 named - osmads1ko (generated in a collaborative study) was characterized and found to display extreme defects in floral organs and an indeterminate FM. Strikingly, in addition to loss of determinacy, FM reverts to a prior developmental fate of inflorescence on whose new rachis are leaf-like malformed florets. We suggest these phenotypes reflect the null phenotype of OsMADS1 and its role in meristem fate maintenance. We tested gene expression levels for some proven targets of OsMADS1 (Khanday et al., 2013) and utilized panicles in two developmental phases- young early FMs (panicles of 0.2 to 0.5 cm) and older florets with organ differentiation (panicles of 0.5 to 1cm). We observed temporally different effects on the regulation of OsMADS34 that together with histology of young osmads1ko inflorescences suggest that the mutant is impeded for spikelet to floral meristem transition. In addition, OsMADS1 had a positive regulatory effect on genes implicated for lemma and palea organ identity such as OsIDS1, OsDH1, OsYABBY1, OsMADS15, OsMADS32, OsDP1 and OsSPL16 in both young and old panicles while OsIG1 was negatively regulated in both phases of development. MADS-box genes important for carpel and ovule development - OsMADS13 and OsMADS58 were had significantly reduced expression in florets undergoing organ differentiation. OsMADS1 positively regulated several other non MADS-box developmental genes - OsSPT, OsHEC2 and OsULT1, whose Arabidopsis homologs control carpel development and FM determinacy. These genes are de-regulated in later stages of osmads1ko floret development and are unaffected in younger panicles. Finally, OsMADS1 continually activated meristem maintenance genes - OsBAM2-like and OsMADS6 while the activation of OSH1 in early floral meristems was later altered to a repressive effect in developing florets. Perhaps such dynamic temporal effects on meristem genes are instrumental in the timely termination of the floral meristem after floral organ differentiation. More importantly, we show that regulation of many of these genes is directly affected by OsMADS1, through our studies on expression levels before and after chemical induction of OsMADS1-GR protein in amiRNAOsMADS1 florets. Further, some key downstream targets were re-affirmed by studying expression status in transgenic lines, with the OsMADS1-EAR repressive protein variant. These results provide new insights into the developmentally phased roles of OsMADS1 on floral meristem regulators and determinants of organ identity to form a determinate rice floret. Gene networks regulated by OsMADS1 during early flower development To identify global targets in early floret meristems, we determined the differential RNA transcriptome in osmads1ko tissues as compared to wild-type tissues. These data revealed regulators of inflorescence architecture, floral organ identity including MADS-box floral homeotic factors, factors for meristem maintenance, auxin response, transport and biosynthesis as some of the important functional classes amongst the 2725 differentially expressed genes (DEGs). Integrating DEGs with OsMADS1 ChIP-seq data (prior studies from our lab) we deciphered direct vs. indirect and positive vs. negatively regulated targets of OsMADS1. These datasets reveal an enrichment for functional categories such as metabolic processes, signaling, RNA transcription and processing, hormone metabolism and protein modification. Using Bio-Tapestry plot as a tool we present a visualization of a floral stage-specific regulatory network for genes with likely functional roles in meristem specification and in organ development. Further, to examine if indirect targets regulated by OsMADS1 could be mediated through transcription factors (that are themselves direct targets), we constructed a small network with the transcription factors OSH1, OSH15 and OsYABBY1 as key nodal genes and we predicted their downstream effects. Taken together, these analyses provide examples of the complex networks that OsMADS1 controls during the process of rice floret development. In summary, we surmise that defect in phytohormone distribution in OsMADS1 knockdown florets results in irregular patterns of lateral organ primordia emergence. In addition, the derangements in the developmentally stage specific expression of floral meristems identity and organ identity genes culminates in miss-specified and irregularly patterned abnormal organs in Osmads1 florets. Thus, our study highlights the versatility of OsMADS1 in regulating components of hormone signaling and response, and its effects on various floral development regulators results in the formation of a single determinate floret on the spikelet. References: Khanday I, Yadav S.R, and Vijayraghavan U. (2013). Plant Physiol 161, 1970–1983. van Mourik S , Kaufmann K, van Dijk AD, Angenent G.C, Merks R.M.H, Molenaar J. (2012). PLOS One 1, e28762 Reinhardt D, Pesce E, Stieger P, Mandel T, Baltensperger K, Bennett M, Traas J, Friml J and Kuhlemeier C. (2003). Nature 426, 255-260 Su Y, Liu Y and Zhang X. (2011) Mol Plant 4, 616–625

A axila foliar em Passiflora L. (Passifloraceae) apresenta uma estrutura complexa: de um mesmo ponto parecem surgir flores e gavinhas, além de uma gema vegetativa também estar presente. A origem da gavinha foi interpretada de diferentes maneiras ao longo da história, sendo considerada desde modificações de um ramo até uma flor. Além disso, a ontogenia dessas estruturas tem início em um único meristema axilar, que geralmente é descrito como capaz de se dividir em dois ou mais meristemas (chamado de \"complexo de gemas\"), cada qual dando origem a uma estrutura diferente (gavinhas e flores). Estudos de expressão gênica demonstram a presença do ortólogo do gene LEAFY de Arabidopsis, em meristemas axilares, florais e de gavinhas, em duas espécies de Passiflora. Esse gene é tipicamente relacionado à transição de fase vegetativa para reprodutiva em diversas angiospermas. Assim, o presente estudo objetivou descrever em detalhes a ontogenia das diferentes estruturas originadas no meristema axilar de diferentes espécies, focando em diferentes fases de vida da planta, bem como averiguar a expressão de ortólogos de APETALA1 (AP1), um gene tipicamente relacionado à identidade de meristemas florais e na determinação de sépalas e pétalas. Como resultado, propomos uma nova interpretação para a ontogenia do complexo de gemas, baseada na produção de brácteas e seus meristemas associados. Demonstramos também que o ortólogo de AP1 se expressa de maneira mais ampla do que aquela encontrada no modelo Arabidopsis, possivelmente desempenhando diversas funções relacionadas à manutenção da indeterminação celular. / The leaf axil in Passiflora L. (Passifloraceae) bears a complex structure: a tendril and one or more flowers seem to arise from the same growing point. In addition, vegetative bud is also present. There are many different interpretations for the origin of the tendril in this group, ranging from modifications of flowers to side shoots. Also, the ontogeny of these structures is often understood as a single meristem which subdivides into a bud complex, comprising the tendril and flower meristems. Recently, the expression of the LEAFY ortholog was demonstrated in the axillary, tendril and floral meristems of two Passiflora species. In Arabidopsis and many angiosperms, this gene is responsible for the shift between vegetative and reproductive phase. Therefore, the present work aimed to describe, in detail, the ontogeny of the bud complex in Passiflora species belonging to different subgenera, including different life stages. The expression of the ortholog of APETALA1, a gene typically related to floral meristem identity and sepal/petal specification was also assessed. As results, we propose a different interpretation for the ontogeny of the bud complex, based on the production of bracts and their associated meristems by the original axillary meristem, which then turns into the tendril meristem. We also demonstrate that expression of AP1 is much broader than that of the Arabidopsis model, and possibly have many other functions related to cell indeterminacy.

Why don’t crop plants grow as fast as they should? In optimal conditions, elite crop varieties routinely outperform those grown in the average field. The vast majority of this reduction in growth activity is due to abiotic stresses such as drought, heat, and nutrient limitation. Abiotic stress reduces plant growth by triggering a reduction of meristem size and causing premature differentiation of proliferating cells. Differentiated cells are no longer able to divide, and smaller meristems have a reduced capacity to restore growth when the abiotic stress passes. We have designed and evaluated a novel high-throughput screening system to identify compounds able to reduce or prevent this premature differentiation in order to retain modest growth capacity in stressful conditions and enable rapid recovery from stress. Such chemicals can be applied to crop plants using existing agricultural methods, and because there is no need for genetic modification, it is widely applicable to many different crop species. Using the novel technique of flow sorting followed by protoplast culture, we have developed a high-throughput automated confocal imaging method to screen chemicals for their effects upon cell differentiation. Meristem protoplasts isolated from the root tips of pROW1:GFP Arabidopsis plants were monitored for differentiation when exposed to different chemicals. To evaluate this system, a library of biologically active small molecules provided by Syngenta was screened against protoplasts and whole plants. Several compounds were identified with the ability to improve Arabidopsis root growth in in vitro growth conditions. Two subsets of these chemicals were identified: a subset of chemicals that improved stress tolerance through modulation of post-meristem differentiation, and a subset of chemicals that improve growth rate by increasing rates of cell division in the root apical meristem. This screening system is able to detect the subset of chemicals that was shown to affect postmeristem differentiation, but not the other subset. No false positives were detected. These results suggest that this single-cell screening system is a powerful, high-throughput method suitable for the detection of molecules for use in crop protection.