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.

This thesis describes the characterization of the Arabidopsis thaliana mutant terminal flower2 (tfl2), the cloning of the corresponding gene, and the analysis of TFL2 function in plant development. The tfl2 mutant is pleiotropic, exhibiting early floral induction in both long and short day conditions, a terminating inflorescence and dwarfing. TFL2 was isolated using a positional cloning strategy, and was found to encode a homolog to HETEROCHROMATIN PROTEIN1 (HP1), previously identified in yeast and animals where it is involved in gene regulation at the level of chromatin, as well as in the structural formation of constitutive heterochromatin. Investigating the light response during seedling photomorphogenesis I found that the tfl2 hypocotyl is hypersensitive to red and far-red light and that tfl2 is impaired in phytochrome mediated light responses such as the shade avoidance response. In the tightly regulated transition to flowering, we have shown that tfl2 might contribute to the interpretation of both external signals such as light and temperature as well as endogenous cues, via FCA, in the autonomous pathway. The Arabidopsis inflorescence meristem is indeterminate, and TFL2 possibly acts to maintain this indeterminate fate by repression of the floral meristem genes APETALA1 and AGAMOUS. In yeast two hybrid experiments TFL2 was shown to interact with IAA5, a protein with suggested functions in auxin regulation. Further, in tfl2 mutants the levels of the auxin indole-3-acetic acid decrease with age in aerial tissues, suggesting a function of TFL2 in regulation of auxin homeostasis and response. In summary, TFL2 contributes to regulation of several aspects of plant development, in accordance with the mutant phenotype and the identity of the TFL2 protein.

The branching or tillering of crops is an important agronomic trait with a major impact on yield. Maintaining an appropriate number of branches allows the plant to use limited light resources and to produce biomass or yield more effectively. The branching process includes the initiation of the axillary meristem leading to bud formation and the further outgrowth of the axillary buds. Phytohormones, including cytokinins and auxin, are known to play major roles in regulating axillary bud outgrowth. Light signals, including light quantity and light quality, are among the most important factors regulating plant growth and are perceived by the action of specialized photoreceptors, including phytochromes. Phytochromes sense red (R) and far-red (FR) light and allow some plants to perceive and respond to competing neighbors by evoking the shade avoidance syndrome (SAS). One component of the SAS is inhibition of branching. Phytochrome B (phyB) is especially important in sensing shade signals and loss of phyB function results in a constitutive shade avoidance phenotype, including reduced branching. While it has been anecdotally reported that phyB-deficient Arabidopsis branches less than wild type, a detailed study of the defects in the process is lacking. In this research, the interactions between light signals, phytochromes and phytohormones in the regulation of branching were assessed using an integrated physiological, molecular and genetic approach.

The diploma thesis deals with the use of meristem cultures in garlic (Allium sativum) breeding. The source material was used variety Tantalum of garlic. The using material, before the isolation of meristem, was tested for the occurrence of viral diseases by immunological tests ELISA (Enzyme-Linked Immuno-Sorbent Assay), also known as EIA (Enzyme Immunoassay). The method used to detect antibodies and antigens. The material was tested for viruses onion yellow dwarf (OYDV - Onion Yellow Dwarf Virus) and virus genus Carlavirus (GCLV - Common Garlic Latent Virus). Then the sound material was sterilized, cultured and then it was obtained meristem. Cultivation of preparation meristem was realised according available methodologies. After preparation meristem put on MS medium with vitamins and growth regulator IAA, NAA and BAP.

La paroi des cellules végétales subit des modifications afin de s'assouplir ou de se rigidifier selon les besoins de la plante. Cette paroi est une structure complexe, composée de cellulose, d'hémicellulose et de pectines. Les modifications subies par les pectines au cours de l'élongation cellulaire sont encore assez peu caractérisées. Dans ce contexte, le but de ce projet est d'étudier le rôle de deux polygalacturonases (PG) dans le développement racinaire de la plante modèle A. thaliana. Les PG sont des enzymes de dégradation des homogalacturonanes (HG), le composant pectique majoritaire de la paroi primaire. Notrehypothèse est que les PG dégradent partiellement les HG des parois longitudinales des cellules racinaires en élongation. Cette dégradation engendrerait un assouplissement pariétal localisé, permettant la croissance anisotropique des cellules. Nos résultats indiquent que les gènes des deux PG étudiées, nommés PG ROOT APICAL MERISTEM (PG RAM) et PG ROOT (PG R) sontexprimés de façon complémentaire dans la racine, l'un dans le méristème racinaire (PG RAM), et l'autre dans la zone d'élongation et de différenciation (PG R). De plus, la sur-expression de la protéine PG R entraine une augmentation de l’élongation des hypocotyles étiolés, ainsi qu'une augmentation de la densité de racines latérales par rapport au sauvage, démontrant son rôle dans le développement racinaire et dans l'allongement cellulaire. Enfin, nous avons démontré que l'expression des gènes de ces PG était contrôlée de façon différentielle par les facteurs de transcription de la famille PLETHORA (PLT). / Plant cell wall structure is modified to control its stiffness or flexibility according to plant’s requirements. The cell wall is a complex structure, composed of cellulose, hemicelluloses and pectins. Pectin modifications during cellular elongation are not very well characterized. In this context, the aim of this project is to study the roles of two polygalacturonases (PG) in the root development on the model plant A. thaliana. PG are homogalacturonans (HG) degradation enzymes, HG being the major pectic component of the primary cell wall. This degradation would lead to a local parietal relaxation, allowing anisotropic growth of the cells. Our results show that the two studied PG, named PG ROOT APICAL MERISTEM (PG RAM) and PG ROOT (PG R), are expressed in complementary areas of the root, either in the root apical meristem (PG RAM) or in the elongated and differenciated root tissues (PG R). Furthermore, the over-expression of PG R results in longer etiolated hypocotyls and increases root density when compared to wild-type, demonstrating its function in root development and in cell elongation. Finally, we demonstrated that expression of these two PG genes is under the control of PLETHORA (PLT) family transcription factors, by differentially ways

Drought is a major environmental stress factor that poses a serious threat to food security. The effects of drought on early reproductive tissue at 1-2 DAP (days after pollination) is irreversible in nature and leads to embryo abortion, directly affecting the grain yield production. We developed a working RNA-Seq pipeline to study maize (Zea mays) drought transcriptome sequenced by Illumina GSIIx technology to compare drought treated and well- watered fertilized ovary (1-2DAP) and basal leaf meristem tissue. The pipeline also identified novel splice junctions - splice variants of previously known gene models and potential novel transcription units. An attempt was also made to exploit the data to understand the drought mediated transcriptional events (e.g. alternative splicing). Gene Ontology (GO) enrichment analysis revealed massive down-regulation of cell division and cell cycle genes in the drought stressed ovary only. Among GO categories related to carbohydrate metabolism, changes in starch and sucrose metabolism-related genes occurred in the ovary, consistent with a decrease in starch levels, and in sucrose transporter function, with no comparable changes occurring in the leaf meristem. ABA-related processes responded positively, but only in the ovaries. GO enrichment analysis also suggested differential responses to drought between the two tissues in categories such as oxidative stress-related and cell cycle events. The data are discussed in the context of the susceptibility of maize kernel to drought stress leading to embryo abortion, and the relative robustness of actively dividing vegetative tissue taken at the same time from the same plant subjected to the same conditions. A hypothesis is formulated, proposing drought-mediated intersecting effects on the expression of invertase genes, glucose signaling (hexokinase 1-dependent and independent), ABA-dependent and independent signaling, antioxidant responses, PCD, phospholipase C effects, and cell cycle related processes. This work was supported by the National Science Foundation Plant Genome Research Pro- gram (grant no. DBI0922747), iPlant Collaborative (NSF DBI-0735191) and also NSF ABI1062472. / Master of Science in Life Sciences

Poaceae (or Gramineae) belong to the grass family and is one of the largest families among flowering plants on land. They include some of the most important cereal crops such as rice (Oryza sativa), barley (Hordeum vulgare), wheat (Triticum aestivum), maize (Zea mays), and sorghum (Sorghum bicolor). The characteristic bushy appearance of grass plants, including cereal crops, is formed by the activities of axillary meristems (AMs) generated in the leaf axil. These give rise to tillers from the basal nodes which recapitulate secondary growth axis and AMs are formed during vegetative development. On transition to flowering the apical meristem transforming to an inflorescence meristem (IM) which produces branches from axillary meristem. These IM gives rise to branches that ultimately bear florets. Vegetative branching/tillering determines plant biomass and influences the number of inflorescences per plant. While inflorescence branching determines the number of florets and hence seeds. Thus the overall activity of axillary meristems plays a key role in determining plant architecture during both vegetative and reproductive stages. In Arabidopsis, research on the plant specific transcription factor LEAFY (LFY) has pioneered our understanding of its regulatory functions during transition from vegetative to reproductive development and its role in specifying a floral meristem (FM) identity to the newly arising lateral meristems. In the FM LFY activates other FM genes and genes for floral organ patterning transcription factors. LFY is strongly expressed throughout the young floral meristems from the earliest stages of specification but is completely absent from the IM (Weigel et al., 1992). LFY expression can also be detected at low levels in the newly emerging leaf primordia during the vegetative phase, and these levels gradually increase until the floral transition (Blazquez et al., 1997; Hempel et al., 1997). In rice, the LFY ortholog-RFL/APO2 is expressed predominantly in very young branching panicles/ inflorescence meristems (Kyozuka et al., 1998; Prasad et al., 2003) while in the vegetative phase RFL is expressed at axils of leaves (Rao et al., 2008). In rice FMs expression is restricted to primordia of lodicules, stamens, carpels and ovules (Ikeda-Kawakatsu et al., 2012). Knockdown of RFL activity or loss of function mutants show delayed flowering and poor panicle branching with reduced number of florets and lower fertility (Rao et al., 2008, Ikeda-Kawakatsu et al., 2012). In some genotypes reduced vegetative axillary branching is also compromised (Rao et al., 2008). On the other hand RFL overexpression leads to the early flowering, attributing a role as an activator for the transition of vegetative meristems to inflorescence meristems (Rao et al., 2008). Thus, RFL shows a distinct developmental expression profile, has unique mutant phenotypes as compared to Arabidopsis LFY thus indicating a divergence in functions. We have used various functional genomics approaches to investigate regulatory networks controlledby RFL in the vegetative axillary meristems and in branching panicles with florets. These regulatory effects influence tillering and panicle branching, thus contributing to rice plant architecture. RFL functions in axillary meristem Vegetative AMs are secondary shoot meristems whose outgrowth determines plant architecture. In rice, AMs form tillers from basal nodes and mutants with altered tillering reveal that an interplay between transcription factors and the phytohormones - auxin, strigolactone underpins this process. We probed the relationship between RFL and other factors that control AM development. Our findings indicate that the derangements in AM development that occur on RFL knockdown arise from its early effects during specification of these meristems and also later effects during their outgrowth of AM as a tiller. Overall, the derailments of both steps of AM development lead to reduced tillering in plants with reduced RFL activity. Our studies on the gene expression status for key transcription factor genes, genes for strigolactone pathway and for auxin transporters gave an insight on the interplay between RFL, LAX1 and strigolactone signalling. Expression levels of LAX1 and CUC genes, that encode transcription factors with AM specification functions, were modulated upon RFL knockdown and on induction of RFL:ΔGR fusion protein. Thus our findings imply a likely, direct activating role for RFL in AM development that acts in part, through attaining appropriate LAX1 expression levels. Our data place meristem specification transcription factors LAX1 and CUC downstream to RFL. Arabidopsis LFY has a predominant role in conferring floral meristem (FM) identity (Weigel et al., 1992; Wagner, 2009; Irish, 2010; Moyroud et al., 2010). Its functions in axillary meristems were not known until recently. The latter functions were uncovered with the new LFYHARA allele with only partial defects in floral meristem identity (Chahtane et al., 2013). This mutant allele showed LFY can promote growth of vegetative AMs through its direct target REGULATOR OF AXILLARY MERISTEMS1 (RAX1), a R2R3 myb domain factor (Chahtane et al., 2013). These functions for Arabidopsis LFY and RAX1 in AMs development are parallel to and redundant with the pathway regulated by LATERAL SUPPRESSOR (LAS) and REGULATOR OF AXILLARY MERISTEM FORMATION1 (ROX1) (Yang et al., 2012; Greb et al., 2003). Interestingly, ROX1 is orthologous to rice LAX1 and our data show LAX1 expression levels in rice panicles and in culms with vegetative AMs is dependent on the expression status of RFL. Thus, we speculate that as compared to Arabidopsis AM development, in rice the LFY-dependent and LFY-independent regulatory pathways for AMs development are closely linked. In Arabidopsis, CUC2 and CUC3 genes in addition to their role in shoot meristem formation and organ separation play a role in AM development possibly by defining a boundary for the emerging AM. These functions for the Arabidopsis CUC genes are routed through their effects on LAS and also by mechanisms independent of LAS (Hibara et al., 2006; Raman et al., 2008). These data show modulation in RFL activity using the inducible RFL:∆GR protein leads to corresponding expression changes in CUC1/CUC2 and CUC3 genes expression in culm tissues. Thus, during rice AM development the meristem functions of RFL and CUC genes are related. Consequent to specification of AM the buds are kept dormant. Bud outgrowth is influenced by auxin and strigolactone signalling pathways. We investigated the transcript levels, in rice culms of genes involved in strigolactone biosynthesis and perception and found the strigolactone biosynthesis gene D10 and hormone perception gene are significantly upregulated in RFL knockdown plants. Further, bioassays were done for strigolactone levels, where we used arbuscular mycorrhiza colonization assay as an indicator for strigolactone levels in wild type plants and in RFL knockdown plants. These data validate higher strigolactone signalling in RFL knockdown plants. To probe the relationship between RFL and the strigolactone pathway we created plants knocked down for both RFL and D3. For comparison of the tillering phenotype of these double knockdown plants we created plants with D3 knockdown alone. We observed reduced tillering in plants with knockdown of both RFL and D3 as compared to the tiller number in plants with knockdown of D3 alone. These data suggest that RFL acts upstream to D3 of control bud outgrowth. As effects of strigolactones are influenced by auxin transport we studied expression of OsPIN1 and OsPIN3 in RFL knockdown plants. Their reduced expression was correlated with auxin deficiency phenotypes of the roots in RFL knockdown plants. These data in conjunction with observations on OsPIN3 the gene expression modulation by the induction of RFL:∆GR allow us to speculate on a relationship between RFL, auxin transport and strigolactones with regard to bud outgrowth. We propose that the low tillering phenotype of RFL knockdown plants arises from weakened PATS, consequent to low levels of PIN1 and PIN3, coupled with moderate increase in strigolactones. Taken together, our findings suggest functions for RFL during AM specification and tiller bud outgrowth. RFL functions in panicle branching Prior studies on phenotypes of RFL knockdown or loss of function mutants suggested roles for RFL in transition to flowering, inflorescence meristem development, emergence of lateral organs and floral organ development (Rao et al., 2008; Ikeda-Kawakatsu et al., 2012). It has been speculated that RFL acts to suppress the transition from inflorescence meristem to floral meristem through its interaction with APO1 (Ikeda-Kawakatsu et al., 2012). The downstream genes regulated by RFL in these processes have not yet been elucidated. To identify direct targets of RFL in developing panicles we adopted ChIP-seq coupled with studies on gene expression modulation on induction of RFL. For the former we raised polyclonal anti-sera and chromatin from branching panicles with few florets. For gene expression modulation studies, we created transgenics with a T-DNA construct where an artificial miRNA against 3’UTR specifically knocked endogenous RFL and the same T-DNA had a second expression cassette for generation of a chemically inducible RFL-ΔGR protein that is not targeted by amiR RFL. Our preliminary ChIP-seq data in the wild type panicle tissues hints that RFL binds to hundreds of loci across the genome thus providing first glimpse of direct targets of RFL in these tissues. These data, while preliminary, were manually curated to identify likely targets that function in flowering, we summarize here some key findings. Our study indicates a role of RFL in flowering transition by activating genes like OsSPL14 and OsPRMT6a. Recent studies indicate that OsSPL14 directly binds to the promoter of OsMADS56 or FTL1, the rice homologs of SOC1 and FT to promote flowering (Lu et al., 2013). As RFL knockdown plants show highly reduced expression of OsMADS50/SOC1 and for RFT1 (Rao et al., 2008), and we show here RFL can bind and induce OsSPL14 expression we suggest the RFL¬OsSPL14 module can contribute to the transition of the SAM to flowering. Further, OsSPL14 in the young panicles directly activates DENSE AND ERECT PANICLE1 (DEP1) to control panicle length (Lu et al., 2013). Thus RFL-OsSPL14-DEP1 module could explain the role of RFL in controlling panicle architecture (Rao et al., 2008; Ikeda-Kawakatsu et al., 2012). Thus RFL plays a role in floral transition and this function is conserved across several LFY homologs. Our data ChIP-seq in the wild type tissue and gene expression modulation studies in transgenics also give molecular evidences for the role of RFL in suppression of floral fate. The direct binding of RFL to OsMADS17, OsYABBY3, OsMADS58 and HD-ZIP-IV loci and the changes in their transcript levels on induction of RFL support this hypothesis. Once the transition from SAM to FM takes place, we speculate RFL represses the conversion of inflorescence branch meristems to floral fate by negatively regulating OsYABBY3, HD-ZIP class IV and OsMADS17 that can promote differentiation. These hypotheses indicate a diverged function for RFL in floral fate repression. Arabidopsis LFY is known to activate the expression of AGAMOUS (AG), whose orthologs in rice are OsMADS3 and OsMADS58. Our studies confirm conservation with regard to RFL binding to cis elements at OsMADS58 locus that is homologous to Arabidopsis AG. But importantly we show altered consequences of this binding on gene expression. We find RFL can suppress the expression of OsMADS58 which we speculate can promote a meristematic fate. Further, we also present the abnormal upregulation of floral organ fate genes on RFL downregulation. These data too indicate functions of RFL, are in part, distinct from the role of Arabidopsis LFY where it works in promoting floral meristem specification and development. These inferences are supported by our data that rice gene homologs for AP1, AP3 and SEP3 are not directly regulated by RFL, unlike their direct regulation by Arabidopsis LFY during flower development. We also report the expression levels of LAX1, FZP, OsIDS1 and OsMADS34 genes involved in meristem phase change and IM branching are RFL dependent. This is consistent with its role in the suppression of determinacy, thereby extending the IM activity for branch formation. But as yet we do not know if these effects are direct. Together, our data report direct targets of RFL that contribute to its functions in meristem regulation, flowering transition, and suppression of floral organ development. Overall, our preliminary data on RFL chromatin occupancy combined with our detailed studies on the modulation of gene expression provides evidence for targets and pathways unique to the rice RFL during inflorescence development. Comparative analysis of genes downstream to RFL in vegetative tillers Vs panicles Tillers and panicle branches arise from the axillary meristems at vegetative and reproductive stages, respectively, of a rice plant and overall contribute to the plant architecture. Some regulatory factors control branching in both these tissues - for example, MOC1 and LAX1. Mutants at these loci affect tillers and panicle branch development thus indicating common mechanisms control lateral branch primordia development (Li et al., 2003; Komatsu et al., 2003; Oikawa and Kyozuka, 2009). Knockdown of RFL activity or loss-of-function mutants cause significantly reduced panicle branching and in few instances, reduction in vegetative axillary branching (Rao et al., 2008; Ikeda- Kawakatsu et al., 2012). We took up the global expression profiling of RFL knockdown plants compared to wild type plants in the axillary meristem and branching panicle tissue. These data provide a useful list of potential targets of RFL in axillary meristem and branching panicle tissue. The comparative analysis of the genes affected in the two tissues indicates only a subset of genes is affected by RFL in both the vegetative axillary meristems and branching panicle. These genes include transcription factors (OsSPL14, Zn finger domain protein, and bHLH domain protein), hormone signalling molecules (GA2 ox9) and cell signalling (LRR protein) as a set of genes activated by RFL in both tissues. On the other hand, these comparative expression profiling studies also show distinct set of genes deregulated by RFL knockdown in these two tissues therefore implicating RFL functions have a tissue-specific context. The genes deregulated only in axillary meristem tissue only include D3- involved in the perception of strigolactone, OsMADS34 speculated to have a role in floral transition and RCN1 involved in transition to flowering. On the other hand, the genes – CUC1, OsMADS3, OsMADS58 involved in organ development and floral meristem determination were found to be deregulated only in panicle tissues of RFL knockdown plants. These data point towards presence of distinct mechanisms for the development of AMs as tillers versus the development of panicle axillary as rachis branches. Overall, these data implicate genes involved in transition to flowering, axillary meristem development and floral meristem development are controlled by RFL in different meristems to thereby control plant architecture and transition to flowering.

[ES] Las leguminosas son un grupo de plantas consideradas de gran importancia por su valor nutricional para la alimentación humana y ganadera. Además, las familias de leguminosas se caracterizan por rasgos distintivos de desarrollo como su inflorescencia compuesta y su compleja ontogenia floral. Para comprender mejor estas características distintivas, es importante estudiar los genes reguladores clave involucrados en el desarrollo de la inflorescencia y la flor. El gen SUPERMAN (SUP) es un factor transcripcional de dedos de zinc (Cys2-Hys2) considerado como un represor activo que controla el número de estambres y carpelos en A. thaliana. Además, SUP está involucrado en la terminación del meristemo floral y el desarrollo de los tejidos derivados del carpelo. El objetivo principal de este trabajo fue la caracterización funcional del ortólogo de SUP en la leguminosa modelo Medicago truncatula (MtSUP). Logramos este objetivo en base a un enfoque de genética reversa, análisis de expresión génica y ensayos de complementación y sobreexpresión. Nuestros resultados muestran que MtSUP es el gen ortólogo de SUP en M. truncatula. MtSUP comparte algunos de los roles ya descritos para SUP con algunas variaciones. Curiosamente, MtSUP controla la determinación del meristemo inflorescente secundario (I2) y de los primordios comunes (CP) a pétalos y estambres. Por tanto, MtSUP controla el número de flores y de pétalos-estambres que producen el meristemo I2 y los primordios comunes, respectivamente. MtSUP muestra funciones novedosas para un gen de tipo SUP, desempeñando papeles clave en los meristemos que confieren complejidad de desarrollo a esta familia de angiospermas. Este trabajo permitió identificar a MtSUP, un gen clave que forma parte de la red reguladora genética que subyace al desarrollo de la inflorescencia compuesta y de las flores en la leguminosa modelo M. truncatula. / [CA] Les lleguminoses són un gran grup de plantes considerades de gran importància pel seu valor nutricional per a l'alimentació humana i ramadera. A més, les famílies de lleguminoses es caracteritzen per trets distintius de desenrotllament com la seua inflorescència composta i la seua complexa ontogènia floral. Per a comprendre millor estes característiques distintives, és important estudiar els gens reguladors clau involucrats en la inflorescència i el desenrotllament floral. El gen SUPERMAN (SUP) és un factor transcripcional de dits de zinc (Cys2-Hys2) considerat com un repressor actiu que controla el nombre d'estams i carpels en A. thaliana. A més, SUP està involucrat en la terminació del meristemo floral i el desenrotllament dels teixits derivats del carpel. "L'objectiu principal d'este treball va ser la caracterització funcional de l'ortòleg de SUP en la lleguminosa model Medicago truncatula (MtSUP) . Aconseguim l'objectiu amb base en un enfocament genètic invers, anàlisi d'expressió gènica i assajos de complementació i sobreexpressió. Els nostres resultats mostren que MtSUP és el gen ortòleg de SUP en M. truncatula. MtSUP compartix alguns dels rols ja descrits per a SUP amb variacions. Curiosament, MtSUP està involucrat en la determinació del meristemo de la inflorescència secundària (I2) i els primordios comuns (CP). Per tant, MtSUP controla el nombre de flors i pètals-estams que produïxen el meristemo I2 i els primordios comuns, respectivament. MtSUP mostra funcions noves per a un gen tipus SUP, exercint papers clau en els meristemos que conferixen complexitat de desenrotllament a esta família d'angiospermes. "Este treball va permetre identificar a MtSUP, un gen clau que forma part de la xarxa reguladora genètica darrere de la inflorescència composta i el desenrotllament de flors en la lleguminosa model M. truncatula. / [EN] Legumes are a large group of plants considered of great importance for their nutritional value in human and livestock nutrition. Besides, legume families are characterized by distinctive developmental traits as their compound inflorescence and complex floral ontogeny. For a better understanding of these distinctive features is important to study key regulatory genes involved in the inflorescence and floral development. The SUPERMAN (SUP) gene is a zinc-finger (Cys2-Hys2) transcriptional factor considered to be an active repressor that controls the number of stamens and carpels in A. thaliana. Moreover, SUP is involved in the floral meristem termination and the development of the carpel marginal derived tissues. The main objective of this work was the functional characterization of the SUP orthologue in the model legume Medicago truncatula (MtSUP). We achieved this objective based on a reverse genetic approach, gene expression analysis, and complementation and overexpression assays. Our results show that MtSUP is the orthologous gene of SUP in M. truncatula. MtSUP shares some of the roles already described for SUP with variations. Interestingly, MtSUP controls the determinacy of the secondary inflorescence (I2) meristem and the common primordia (CP). Thus, MtSUP controls the number of flowers and petal-stamens produced by the I2 meristem and the common primordia respectively. MtSUP displays novel functions for a SUP-like gene, playing key roles in the meristems that confer developmental complexity to this angiosperm family. This work allowed to identify MtSUP, a key gene that participates in the genetic regulatory network underlying compound inflorescence and flower development in the model legume M. truncatula. / I would like to thanks the Spanish Ministry of Economy and Competitiveness for the grant (MINECO; BIO2016-75485-R) that supported this work. Special thanks to the Generalitat Valenciana for funding my doctorate with the Santiago Grisolía predoctoral scholarships / Rodas Méndez, AL. (2021). MtSUPERMAN controls the number of flowers per inflorescence and floral organs in the inner three whorls of Medicago truncatula [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/171474

O florescimento é um processo chave no desenvolvimento vegetal. A mudança de identidade do meristema apical de vegetativo para reprodutivo desencadeia reprogramação genética com efeitos em todo o corpo vegetal. Arabidopsis thaliana é conhecida como o principal modelo de estudo para esse processo apresentando até o momento cinco principais vias genéticas regulatórias. Tais vias apresentam redundância, sendo complexa a eliminação total da transição de fase nessa espécie. A via AGE, regulada pela idade da planta, tem como principais reguladores o mir156 e seus alvos diretos, os fatores de transcrição da família SPL/SBP (SQUAMOSA PROMOTER BINDING PROTEIN-like). Uma segunda via é controlada pelo fitohormônio giberelina (GA), o qual atua de maneira oposta em Arabidopsis thaliana (arabidopsis) e Solanum lycopersicum L. (tomateiro). Em tomateiro, diferentemente de arabidopsis, o cruzamento entre mutantes com conteúdo alterado de GA e plantas transgênicas superexpressando o miR156 (156OE; SILVA et al., 2014) demonstraram efeito sinérgico no atraso do tempo de florescimento. A aplicação de GA3 em plantas 156OE apresenta efeito similar aos cruzamentos citados sobre a transição do meristema apical. Em um dos cruzamentos entre mutantes da via GA e plantas 156OE, foi possível obter plantas apresentando completo bloqueio da transição de fase vegetativo-reprodutivo. A oferta extra do florígeno SINGLE FLOWER TRUSS (SFT) via enxertia não foi suficiente para restaurar a transição de fase nessas plantas, sugerindo que vias associadas à GA e AGE regulam alvos em comum, os quais podem ser independentes da regulação por SFT. Além disso, a regulação transcricional, e possivelmente pós-transcricional de alguns genes SBPs por diferentes vias associadas à GA, sugere uma complexa inter-relação entre as vias GA e AGE em tomateiro durante o florescimento. A ação combinada das vias GA e AGE foi capaz de inibir completamente o florescimento em tomateiro, regulação oposta ao verificado na planta modelo Arabidopsis thaliana. O efeito inibitório de GA sobre o florescimento é também visualizado em plantas lenhosas, sugerindo que as descobertas científicas realizadas em tomateiro podem ser expandidas para essas espécies, nas quais a experimentação é lenta e laboriosa / The flowering process is a major developmental event during the plant life cicle. The meristem identity switches from vegetative to reproductive, triggering substantial genetic modifications that affect the whole plant body. Arabidopsis thaliana is a major model for flowering with five different pathways controlling this process. These pathways are redundant, making complex the complete elimination of phase change in this species. One of the pathways is termed AGE since it is regulated by the time of development. The miR156 and its direct target SBP (SQUAMOSA PROMOTER BINDING PROTEIN-like) are the main regulators of the AGE pathway. A second pathway is controlled by the phytohormone gibberellin (GA), which acts in opposite ways when comparing Arabidopsis thaliana and tomato. In tomato, unlike Arabidopsis, the cross between mutants with altered contents of GA and transgenic plants overexpressing the miR156 (156OE; SILVA et al, 2014) showed synergistic effect in delayed flowering time. Treatments of GA3 in plants 156OE lead to similar effects visualized on the crosses above related to meristem transition. Among the crosses between GA mutants and 156OE plants, one double mutant could completely abolish the phase change in tomato. An extra offer of the florigen (SINGLE FLOWER TRUSS or SFT) by grafting experiments was unable to restore the flowering process in this double mutant. It suggests, pathways associated to GA and AGE regulate common downstream targets, which could be independent of SFT regulation. Moreover, the transcriptional regulation, and possible the post-transcriptionally regulation of some SBP targets by different pathways associated to GA, suggest a complex network between GA and AGE during the flowering in tomato. The combined action of GA and AGE pathways can complete impaired the flowering in tomato, this interaction is opposed to the model Arabidopsis thaliana. The negative effect of GA over the time of flowering is presented in wood plants, suggesting the scientific discoveries in tomato could be expanded to these species, which experiments are slow and laborious

Les plantes de la famille des légumineuses ont la particularité d’héberger intracellulairement des bactéries du sol communément appelées rhizobia. Cette interaction symbiotique se déroule au sein de la nodosité, un organe formé de-novo au niveau racinaire. L’activité nitrogénase bactérienne y permet la réduction de l’azote atmosphérique en NH3 assimilable par la plante. Si les mécanismes moléculaires gouvernant la reconnaissance entre les deux partenaires, l’infection intracellulaire et l’organogénèse des nodosités ont été particulièrement bien décrits au cours des dernières décennies ; peu d’informations sont quant à elles disponibles sur l’origine de ce programme morphogénétique nouveau chez les Angiospermes. Les nodosités des deux légumineuses modèles Medicago truncatula et Pisum sativum sont qualifiées d’indéterminées en raison de la persistance d’un méristème en position apicale. Les nodosités des mutants noot (nodule-root) chez M. truncatula et coch (cochleata) chez le pois développent des racines ectopiques à partir des tissus vasculaires des nodosités, montrant ainsi que les nodosités et racines sont plus apparentées que leur simple comparaison anatomique ne pouvait le suggérer. En outre, l‘activité mérsitématique des nodosités est fortement perturbée chez ces deux mutants qui présentent des nodosités multilobées et élargies. Nous avons montré que les gènes MtNOOT et PsCOCHLEATA étaient orthologues aux gènes AtBLADE-ON-PETIOLE1 et 2 qui codent deux activateurs transcriptionels redondants et cruciaux pour la régulation de nombreux processus développementaux chez Arabidopsis thaliana. En raison de la forte conservation des fonctions biologiques des protéines NOOT, BOPs et COCH, notamment pour la régulation de la morphologie foliaire et florale, de l’architecture de l’inflorescence et de la formation des zones d’abscission, nous proposons que ces fonctions représentent les fonctions ancestrales de la famille des gènes NBCL (NOOT BOP COCH LIKE). L’étude de déterminants hormonaux et génétiques du méristème racinaire dans les nodosités sauvages et mutantes noot ainsi que la caractérisation de l’homéose nodule/racine nous ont permis de dégager des parallèles importants entre les tissus périphériques de la nodosité et ceux de la racine. Nous proposons donc un modèle de développement des tissus vasculaires de la nodosité par co-option du programme racinaire dont la répression est en partie assurée par NOOT. / Legume plants are able to house intracellularly soil bacteria collectively called rhizobia. This symbiotic process takes place in a new organ generally formed on the host roots, the nodule. This interaction allows atmospheric nitrogen fixation to the benefit of the plant by using the bacterial nitrogenase activity. Despite an exhaustive description of molecular determinants of this interaction allowing partners recognition, intracellular accommodation and early nodule organogenesis, less is known about cell lineage and identity of the nodule morphogenetic pathway which is thought to represent a recent acquisition during Angiosperms evolution. Nodules from model legumes such as Medicago truncatula or Pisum sativum are described as indeterminate because of the persistence of a distal meristem. The noot (nodule-root) and coch (coch) mutants, in M. truncatula and P. sativum respectively, develop ectopic roots from the nodule vasculature, suggesting that roots and symbiotic nodules are more closely related than previously admitted based on their anatomical comparison. Moreover, the meristematic activity is strongly modified in noot and coch nodules that harbor numerous and enlarged lobes. We showed that NOOT and COCH are orthologs to AtBLADE-ON-PETIOLE1 and 2 redundant transcriptional activators that represent key regulators of versatile plant developmental processes in Arabidopsis thaliana. Because of the conservation of biological functions controlled by NOOT, BOPs and COCH proteins, in particular the regulation of leaf and floral morphologies, abscission zones formation and inflorescence architecture, we proposed that such functions are inherited from a NBCLs (NOOT BOP COCH LIKE) ancestral gene. Our studies of hormonal and genetic determinants of the root meristem in noot and wild-type nodules as well as the characterization of nodule-to-root homeosis have highlighted important parallels between nodule peripheral tissues and roots. We thus propose a model of nodule vascular unit maintenance by the NOOT-dependent repression of a co-opted root morphogenetic program.