Plant architecture is an important agronomic trait driven by meristematic activities. Indeterminate meristems set repeating phytomers while determinate meristems produce terminal structures. The centroradialis/terminal flower1/self pruning (CETS) gene family modulates architecture by controlling determinate and indeterminate growth. Cotton (G. hirsutum) is naturally a photoperiodic perennial cultivated as a day-neutral annual. Management of this fiber crop is complicated by continued vegetative growth and asynchronous fruit set. Here, cotton CETS genes are phylogenetically and functionally characterized. We identified eight CETS genes in diploid cotton (G. raimondii and G. arboreum) and sixteen in tetraploid G. hirsutum that grouped within the three generally accepted CETS clades: flowering locus T (FT)-like, terminal flower1/self pruning (TFL1/SP)-like, and mother of FT and TFL1 (MFT)-like. Over-expression of single flower truss (GhSFT), the ortholog to Arabidopsis FT, accelerates the onset of flowering in Arabidopsis Col-0. In mutant rescue analysis, this gene driven by its native promoter rescues the ft-10 late flowering phenotype. GhSFT upstream sequence was used to drive expression of the uidA reporter gene. As anticipated, GUS accumulated in the vasculature of Arabidopsis leaves. Cotton has five TFL1-like genes, all of which delay flowering when ectopically expressed in Arabidopsis; the strongest phenotypes fail to produce functional flowers. Three of these genes, GhSP, GhTFL1-L2, and GhBFT-L2, rescue the early flowering tfl1-14 mutant phenotype. GhSPpro:uidA promoted GUS activity specifically in plant meristems; whereas, other GhTFL1-like promoters predominately drove GUS activities in plant vascular tissues. Finally, analysis of Gossypium CETS promoter sequences predicted that GhSFT, GhSP, GhTFL1-L1, GhTFL1-L2 and GhBFT-L2 are regulated by transcription factors involved in shoot and flowering development. Analysis of cotton's two MFT homologs indicated that neither gene functions to control shoot architecture. Our results emphasize the functional conservation of members of this gene family in flowering plants and also suggest this family as targets during artificial selection of domestication.

A large sector of the forestry industry of South Africa comprises Eucalyptus species, covering approximately 49% of the forestry plantation area. Polyploidy induction has become an attractive tool to increase yield and reduce invasiveness in forestry species. Polyploidy induction in Eucalyptus using colchicine treatments on seed and axillary buds was undertaken to produce tetraploids that could be used in breeding programmes; specifically to increase yield and decrease species invasiveness through the production of triploids after crossing with diploid parents. Eight seedlots of E. urophylla and seven of E. grandis were treated with four colchicine concentrations (0.00, 0.01, 0.03, 0.05%) at two exposure times (18 h and 24 h), treating two seeds per treatment, repeated eight times. For axillary bud induction, 20 buds of two E. grandis clones and three E. grandis × E. urophylla hybrids and one E. grandis × E. nitens hybrid were treated with four colchicine concentrations (0.0, 0.5, 1.0, 1.5%) for three consecutive days. A known tetraploid hybrid E. grandis E. camaldulensis and its corresponding diploid were included as reference material. Seedlings and bud sports were pre-screened by determining stomatal guard cell lengths. Seedlings and bud sports displaying cell lengths significantly (p<0.0001) larger than the diploid were selected as putative polyploids. Polyploidy was then confirmed by quantifying the DNA content using flow cytometry. Stomatal frequencies and guard cell chloroplast frequencies were also determined in the induced tetraploid seedlings to evaluate their suitability to discern between ploids. All putative polyploidy seedlings, identified in the pre-screening process, were confirmed, using flow cytometry, as either tetraploids or mixoploids. Of the 17 E. urophylla putative polyploids, from various seedlots, six were tetraploid and 11 mixoploid. In E. grandis one of the five putative polyploids, from various seedlots, was tetraploid and four mixoploid. Pre-screening of bud sports was less accurate; only four of the 12 E. grandis hybrid putative polyploids were mixoploid and only three of the six E. grandis putative polyploids were mixoploid. E. urophylla seedlings were more sensitive to colchicine than E. grandis seedlings displaying a lower survival rate (52%) than E. grandis (63%). Extreme treatments that caused the lowest survival rates were also responsible for most of the polyploidy successful inductions; 0.05%/18 h and 0.05%/24 h for E. urophylla and 0.03%/24 h and 0.05%/24 h for E. grandis. Phenotypic effects of colchicine included shorter, thicker roots and hypocotyls; darker leaves; longer and narrower leaves in some tetraploids; and asymmetrical leaf margins in many mixoploids and tetraploids compared with the controls. In the tetraploids, stomata were significantly larger (p<0.0001) and less frequent (p<0.001). A significant (p<0.001) increase in the number stomatal chloroplasts was also ascertained. Confirmed mixoploid seedlings all displayed tetraploid leaves based on stomatal size and thus classified as periclinal chimeras. In bud sports, only leaves with islands of diploid and tetraploid stomata in the confirmed mixoploids were encountered. Mixoploid bud sports were thus either sectional or mericlinal chimeras. Stomatal size proved to be a suitable pre-screening method, especially in polyploidy induction in seedlings. Additionally confirmed tetraploids exhibited significantly different stomatal frequencies and stomatal chloroplast frequencies compared with the diploids, thus proving to be suitable detection methods for polyploidy screenings. Polyploidy induction in seed was effective, however, less effective in axillary buds which requires further research to refine methods. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2008.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2008. / Härtill 4 uppsatser.

A cana-de-açúcar é uma das principais culturas agroindustriais do Brasil. Como outras culturas de importância econômica, é também afetada por diversos patógenos como fungos, bactérias, vírus e fitoplasmas que podem limitar sua produção. A bactéria fastidiosa Leifsonia xyli subsp. xyli (Lxx) é o agente causal do raquitismo-da-soqueira da cana-de-açúcar (RSD). Esta doença não apresenta sintomas externos característicos de fácil reconhecimento. Dessa forma, a incidência do RSD pode não ser perceptível durante inspeções visuais de campo e, assim, ser facilmente disseminado de uma região para outra. A bactéria restrita ao xilema é transmitida mecanicamente de plantas infectadas para plantas saudáveis por meio da seiva presente em ferramentas de corte e em outros equipamentos durante o plantio, o cultivo e a colheita da cultura. Em razão da importância da cana-de-açúcar e do dano causado por Lxx, este trabalho visou avaliar a termoterapia e o cultivo in vitro em dois experimentos direcionados à obtenção de plantas de cana-de-açúcar da variedade SP80-3280 livres de Leifsonia xyli subsp. xyli e, assim, fornecer subsídio para o aprimoramento das medidas atuais de controle. O primeiro experimento combinou a termoterapia de toletes de uma gema (52 °C por 30 minutos) com o cultivo de meristemas apicais de três tamanhos (0,5 mm, 1,0 mm e 1,5 mm); o segundo combinou três tempos de termoterapia (1 hora, 2 horas e 3 horas) a 50 °C em gemas laterais imaturas isoladas do tolete com o cultivo in vitro. No primeiro experimento, os resultados de RT-PCR das amostras coletadas aos 15 dias na casa de vegetação mostraram que os três tamanhos de meristemas produziram plantas com títulos bacterianos em média 25 vezes mais baixos que suas genitoras, mas não as isentaram completamente de Lxx, com exceção de 8 plantas que não apresentaram Lxx com 90 dias de cultivo (4 plantas provenientes do cultivo de meristemas de 0,5 mm, 1 planta de meristema de 1,0 mm e 3 plantas de meristemas de 1,5 mm). Além disso, os meristemas de 0,5 mm não necessariamente regeneraram plantas com títulos mais baixos da bactéria quando comparados com os meristemas de 1,0 mm e 1,5 mm, mostrando que estes tamanhos testados não apresentaram correlação direta com o nível de infecção da planta regenerada por eles. Em relação ao segundo experimento, os resultados de RT-PCR mostraram que o incremento no tempo de termoterapia proporcionou maior redução nos títulos bacterianos, sendo os tratamentos de 2 horas e 3 horas os mais bem sucedidos com média de redução de 80,08% e 81,73%, respectivamente, mas não foi capaz de eliminar a bactéria. Portanto, estes experimentos demonstram que a termoterapia associada ao cultivo de tecidos é uma metodologia promissora, pois apresentaram redução da presença da bactéria Lxx em plantas de cana-de-açúcar, mas por não terem sido totalmente efetivos na eliminação da mesma, reforça a necessidade de estudos complementares. / Sugarcane is one of the major agro-industrial crops of Brazil. Like other crops of economic importance, it\'s also affected by several pathogens such as fungus, bacteria, viruses and phytoplasmas that may limit its production. The fastidious bacterium Leifsonia xyli subsp. xyli (Lxx) is the causal agent of ratoon stunting disease in sugarcane (RSD). This disease shows no external symptoms characteristic of easy recognition. Thus, the incidence of RSD may not be noticeable during visual inspection in the field and be easily spread from one region to another. The xylem-limited bacterium is transmitted mechanically from infected plants to healthy plants by the sap present in cutting tools and others equipments for planting, growing and harvesting the crop. Due to the importance of sugarcane and the damage caused by Lxx, this work aimed to evaluate thermotherapy and in vitro culture in two experiments in order to obtain sugarcane plants of variety SP80-3280 free from Leifsonia xyli subsp. xyli and thus provide subsidy for the improvement of current control methods. The first experiment combined thermotherapy on the individual bud (52 °C for 30 minutes) and the cultivation of apical meristems of three sizes (0,5 mm, 1,0 mm and 1,5 mm); the second combined three period of thermotherapy (1 hour, 2 hours and 3 hours) at 50 °C in immature lateral buds totally isolated from the tissue source followed by in vitro culture. In the first experiment, the results of RT-PCR samples collected at 15 days in the greenhouse showed that the three sizes of meristems produced plants with bacterial titers on average 25 times lower than their mother plants, but not completely exempted them from Lxx, except 8 plants that showed no Lxx after 90 days of cultivation (4 plants were from cultivation of meristems with 0,5 mm, 1 plant from 1,0 mm meristem and 3 plants from 1,5 mm meristems size). Furthermore, the meristems of 0,5 mm does not necessarily regenerate plants with lower title of the bacteria compared with the meristems of 1.0 mm and 1.5 mm, showing that these sizes tested did not present direct correlation with the level of infection on the plants regenerated from them. Regarding the second experiment, RT-PCR results showed that the increase in time of thermotherapy provided greater reduction in bacterial titers, and the treatments of 2 and 3 hours were the most successful with average reduction of 80,08 % and 81,73%, respectively, but was not able to eliminate the bacteria. Therefore, these experiments showed that thermotherapy associated with tissue culture is a promising methodology because it reduced titer of Lxx in sugarcane, but were not fully effective in eliminating the same, reinforcing the need for complementary studies.

Les organes aériens des plantes sont générés par le méristème apical caulinaire (MAC), dont la mise en place et le maintien ont été largement étudiés. Alors qu’un vaste réseau de gènes assure une régulation robuste de la population de cellules souches, deux gènes se distinguent ; CLAVATA3 (CLV3) et WUSCHEL (WUS). CLV3 s’exprime dans les cellules souches et code pour un peptide signal dont la liaison à des récepteurs transmembranaires mène à la sous-régulation de WUS. Ce dernier code pour un facteur de transcription dans le centre organisateur sous-jacent. En retour, WUS active directement l’expression de CLV3 et l’équilibre entre ces deux molécules est primordial pour la restriction de la population de cellules souches. La perte d’activité de CLV3 conduit à une augmentation de la taille du MAC, tandis que la perte d’activité de WUS abolit le MAC. Selon le modèle actuel, l’apex élargi des mutants clv3 est composé de cellules souches en sur-prolifération. Précédemment, notre groupe a couplé la microscopie à force atomique (mesurant la rigidité cellulaire) à la microscopie confocale (déterminant l’identité cellulaire) et a montré que l’identité des cellules souches est corrélée à une rigidité plus élevée. Dans cette thèse, je montre que les MAC clv3 ont des défauts d’organisation et de mécanique puisque leurs cellules sont moins rigides que ce que prédit le modèle, suggérant que les MAC clv3 diffèrent mécaniquement des cellules souches. J’examine cette contradiction en utilisant un ensemble de gènes exprimés dans différents domaines du MAC pour montrer que les MAC clv3 sont des mosaïques de cellules exprimant simultanément des gènes indiquant un état indifférencié et d’autres indiquant des états de différenciation. De plus, je montre que la composition cellulaire du MAC clv3 diffère de celle du sauvage, que la taille des cellules est dérégulée et que la surface du MAC clv3 est altérée.Notre hypothèse est que les cellules des MAC clv3 subissent un phénomène de ‘stop-start’, au cours duquel leur identité oscille entre cellule souche et cellule différenciée, conduisant à des changements morphométriques à l’origine des phénotypes clv3. En résumé, le ré-examen du rôle que joue CLV3 dans la morphogenèse au niveau du MAC, et donc du modèle CLV-WUS d’homéostasie des cellules souches, me mène à la conclusion que notre vision actuelle est limitée et que les paramètres mécaniques sont à prendre en compte pour une compréhension plus exhaustive des cellules souches. / The shoot apical meristem (SAM) gives rise to above-ground organs and its establishment and homeostasis have been extensively studied. While a vast genetic network ensures the robust regulation of the stem cell population, two genes, CLAVATA3 (CLV3) and WUSCHEL (WUS), are key players. CLV3 is expressed in stem cells and encodes a secreted peptide to signal via transmembrane receptors to downregulate WUS, which encodes a transcription factor in the underlying organising centre. In turn, WUS directly activates the expression of CLV3 and the balance between the two molecules restrains the stem cell pool. The loss of CLV3 activity leads to an increase in SAM size, whereas the loss of WUS activity abolishes the SAM. The prevailing model is that the enlarged clv3 apex is composed of over-proliferating stem cells.Previously, our group coupled atomic force microscopy (to measure cell rigidity) and confocal microscopy (to determine cell identity) to show that stem cell identity correlates with increased stiffness. In this thesis, I show that in addition to altered mechanics, enlarged clv3 SAM also display severe defects in cell organisation. I find that cells in clv3 SAM are soft, instead of being stiff, as we had predicted in light of the model regarding the clv3 phenotype. Our data instead suggest that clv3 SAM differ mechanically from stem cells. I further investigate this contradiction using genetic markers for different domains of the SAM and show that clv3 SAM are in fact mosaic structures, made up of cells that simultaneously express genes that indicate an undifferentiated state and several that indicate multiple states of differentiation. Additionally, I show that the cellular makeup of mutant SAM is significantly altered from the wild type, with a misregulation of cell size in the outer cell layers. Furthermore, mutant SAM also display altered surface smoothness from wild-type SAM.Our working hypothesis is that in clv3 mutant SAM, cells undergo a constant stop-start phenomenon, where they cycle between stemness and specification, resulting in cell-level morphometric changes that generate the characteristic clv3 phenotypes. In summary, during my thesis, I have re-examined the role of CLV3 in morphogenesis at the SAM, and thus the CLV-WUS model of stem cell homeostasis. I conclude that the existing view in the field is limited, and that mechanical parameters need to be considered for a fuller understanding of stem cells.

A cana-de-açúcar é uma das principais culturas agroindustriais do Brasil. Como outras culturas de importância econômica, é também afetada por diversos patógenos como fungos, bactérias, vírus e fitoplasmas que podem limitar sua produção. A bactéria fastidiosa Leifsonia xyli subsp. xyli (Lxx) é o agente causal do raquitismo-da-soqueira da cana-de-açúcar (RSD). Esta doença não apresenta sintomas externos característicos de fácil reconhecimento. Dessa forma, a incidência do RSD pode não ser perceptível durante inspeções visuais de campo e, assim, ser facilmente disseminado de uma região para outra. A bactéria restrita ao xilema é transmitida mecanicamente de plantas infectadas para plantas saudáveis por meio da seiva presente em ferramentas de corte e em outros equipamentos durante o plantio, o cultivo e a colheita da cultura. Em razão da importância da cana-de-açúcar e do dano causado por Lxx, este trabalho visou avaliar a termoterapia e o cultivo in vitro em dois experimentos direcionados à obtenção de plantas de cana-de-açúcar da variedade SP80-3280 livres de Leifsonia xyli subsp. xyli e, assim, fornecer subsídio para o aprimoramento das medidas atuais de controle. O primeiro experimento combinou a termoterapia de toletes de uma gema (52 °C por 30 minutos) com o cultivo de meristemas apicais de três tamanhos (0,5 mm, 1,0 mm e 1,5 mm); o segundo combinou três tempos de termoterapia (1 hora, 2 horas e 3 horas) a 50 °C em gemas laterais imaturas isoladas do tolete com o cultivo in vitro. No primeiro experimento, os resultados de RT-PCR das amostras coletadas aos 15 dias na casa de vegetação mostraram que os três tamanhos de meristemas produziram plantas com títulos bacterianos em média 25 vezes mais baixos que suas genitoras, mas não as isentaram completamente de Lxx, com exceção de 8 plantas que não apresentaram Lxx com 90 dias de cultivo (4 plantas provenientes do cultivo de meristemas de 0,5 mm, 1 planta de meristema de 1,0 mm e 3 plantas de meristemas de 1,5 mm). Além disso, os meristemas de 0,5 mm não necessariamente regeneraram plantas com títulos mais baixos da bactéria quando comparados com os meristemas de 1,0 mm e 1,5 mm, mostrando que estes tamanhos testados não apresentaram correlação direta com o nível de infecção da planta regenerada por eles. Em relação ao segundo experimento, os resultados de RT-PCR mostraram que o incremento no tempo de termoterapia proporcionou maior redução nos títulos bacterianos, sendo os tratamentos de 2 horas e 3 horas os mais bem sucedidos com média de redução de 80,08% e 81,73%, respectivamente, mas não foi capaz de eliminar a bactéria. Portanto, estes experimentos demonstram que a termoterapia associada ao cultivo de tecidos é uma metodologia promissora, pois apresentaram redução da presença da bactéria Lxx em plantas de cana-de-açúcar, mas por não terem sido totalmente efetivos na eliminação da mesma, reforça a necessidade de estudos complementares. / Sugarcane is one of the major agro-industrial crops of Brazil. Like other crops of economic importance, it\'s also affected by several pathogens such as fungus, bacteria, viruses and phytoplasmas that may limit its production. The fastidious bacterium Leifsonia xyli subsp. xyli (Lxx) is the causal agent of ratoon stunting disease in sugarcane (RSD). This disease shows no external symptoms characteristic of easy recognition. Thus, the incidence of RSD may not be noticeable during visual inspection in the field and be easily spread from one region to another. The xylem-limited bacterium is transmitted mechanically from infected plants to healthy plants by the sap present in cutting tools and others equipments for planting, growing and harvesting the crop. Due to the importance of sugarcane and the damage caused by Lxx, this work aimed to evaluate thermotherapy and in vitro culture in two experiments in order to obtain sugarcane plants of variety SP80-3280 free from Leifsonia xyli subsp. xyli and thus provide subsidy for the improvement of current control methods. The first experiment combined thermotherapy on the individual bud (52 °C for 30 minutes) and the cultivation of apical meristems of three sizes (0,5 mm, 1,0 mm and 1,5 mm); the second combined three period of thermotherapy (1 hour, 2 hours and 3 hours) at 50 °C in immature lateral buds totally isolated from the tissue source followed by in vitro culture. In the first experiment, the results of RT-PCR samples collected at 15 days in the greenhouse showed that the three sizes of meristems produced plants with bacterial titers on average 25 times lower than their mother plants, but not completely exempted them from Lxx, except 8 plants that showed no Lxx after 90 days of cultivation (4 plants were from cultivation of meristems with 0,5 mm, 1 plant from 1,0 mm meristem and 3 plants from 1,5 mm meristems size). Furthermore, the meristems of 0,5 mm does not necessarily regenerate plants with lower title of the bacteria compared with the meristems of 1.0 mm and 1.5 mm, showing that these sizes tested did not present direct correlation with the level of infection on the plants regenerated from them. Regarding the second experiment, RT-PCR results showed that the increase in time of thermotherapy provided greater reduction in bacterial titers, and the treatments of 2 and 3 hours were the most successful with average reduction of 80,08 % and 81,73%, respectively, but was not able to eliminate the bacteria. Therefore, these experiments showed that thermotherapy associated with tissue culture is a promising methodology because it reduced titer of Lxx in sugarcane, but were not fully effective in eliminating the same, reinforcing the need for complementary studies.

Tillers are vegetative branches found in grasses, which develop in early stages of plant life. Located at the base of the central stalk, tillers have agronomical importance by increasing seed production with fewer tillers, or providing alternative forms of biofuel with more tillers. As grains have typically decreased tiller number while undergoing domestication, we explored wild and domesticated strains of varying grains by doing a morphological analysis on tiller development. This thesis shows how the decrease of tillers through in domestication cereals shows diversity not only across maize, Sorghum, and Setaria, but also between lines of maize and Setaria species. To do so, we first measured axillary bud growth across these grasses and compared bud initiation, growth, dormancy and outgrowth. While maize inbred B73 demonstrated a tiller dormancy pattern by initiating buds, growing buds and then bud dormancy we measured growth in Sorghum and Setaria to compare and found that although Sorghum patterns dormancy similar to maize, Setaria had more than one way tiller suppression not previously expected. We look further at Setaria buds with a statistical analysis of tiller origin and bud frequency in a wild strain and two domesticated strains of Setaria. Furthermore we performed Scanning Electron Microscopy (SEM) to have a clear understanding of bud initiation or lack of initiation in Setaria italica (B100) comparing it to its wild ancestor Setaria viridis. Because of the diversity in Setaria, we re-visited maize tiller domestication by taking bud measurements, performing SEMs and counting bud frequency on other strains of inbred maize. We found that maize also shows diversity in its patterning of tiller domestication. These results demonstrate that there is diversity in the patterns in which tiller domestication has occurred. This diversity is shown here through differences in tiller bud decisions to initiate or not initiate, or to have axillary buds go dormant post-initiation. Furthermore this variance is shown through differences in bud frequency counts, growth measurements, SEMs, and where tiller branches originate across the grains of maize, Sorghum and Setaria.

A família PUF é um conjunto de proteínas que se ligam a mRNA regulando sua estabilidade e tradução em processos chave do desenvolvimento. Entre as 25 proteínas de Arabidopsis contendo as repetições PUF, três delas, APUM-I, APUM-2 e APUM-3, apresentam ~90% de identidade e colocalizam temporal e espacialmente nos meristemas apical e axilares de caule, zona de elongação da raiz e no periciclo durante a formação de calos e de raízes laterais, além de estames e polens. Ensaios de RT-PCR mostraram que a relação de expressão entre eles é a mesma em todos os órgãos analisados. Além disso, plantas nocautes apum-1- e apum-2- não apresentam fenótipo alterado, sugerindo redundância funcional. Plantas com a expressão dessas proteínas afetadas por RNA antisense apresentaram folhas cloróticas e reduzidas, raízes mais curtas e menos ramificadas e baixa fertilidade, fenótipo semelhante ao de plantas que superexpressam KRP-2, um inibidor de CDK. O transcrito KRP-2 apresenta um elemento de ligação AraPum no 3\'-UTR sugerindo ser um possível alvo para APUM. Em adição, plantas antisense têm aumento de transcritos KRP-2 em relação a selvagens. Assim, foi proposto que essas proteínas agem coordenando a formação de folhas e raízes pela influência na tradução de KRP-2. A função ancestral das proteínas PUF de manter o ciclo celular em detrimento da diferenciação, parece ser conservada em plantas. / The PUF family is a group of conserved proteins that bind to rnRNAs regulating its stability and translation in key developrnental processes. Among the twenty five Arabidopsis proteins with PUF repeats, we found that three highly similar members, APUM-I, APUM-2 and APUM-3 (~90% identity) and co-localize spatially and temporally in the shoot apical and axillaries meristems, root elongation zone and pericycle during callus and lateral root formation, as well as stamens and pollens. RTPCR assays showed that these proteins have similar expression profiles in ali organs analyzed. Moreover, plant apum-1 and apum-2 knockouts have no detectably altered phenotype, suggesting a functional redundancy between them. Plants in which APUM-I, APUM-2 and APUM-3 expression were reduced through antisense RNA, showed chlorotic and reduced leaves, shorter and less ramificated roots and low fertility. This phenotype is similar to that of plants over-expressing the KRP-2 gene, a CDK inhibitor. An AraPum binding element at 3\'-UTR of the KRP-2 transcript suggests that it may be a possible target for APUM. In addition, in comparison to wild-type plants, antisense plants have increased KRP-2 transcripts levels. We proposed that APUM proteins act by coordinating leaf and root formation by way of influencing KRP-2 transiation. The ancestral function of PUF proteins in the maintenance of the cell cycle, to detriment of differentiation, seems to be conserved in plants.

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 família PUF é um conjunto de proteínas que se ligam a mRNA regulando sua estabilidade e tradução em processos chave do desenvolvimento. Entre as 25 proteínas de Arabidopsis contendo as repetições PUF, três delas, APUM-I, APUM-2 e APUM-3, apresentam ~90% de identidade e colocalizam temporal e espacialmente nos meristemas apical e axilares de caule, zona de elongação da raiz e no periciclo durante a formação de calos e de raízes laterais, além de estames e polens. Ensaios de RT-PCR mostraram que a relação de expressão entre eles é a mesma em todos os órgãos analisados. Além disso, plantas nocautes apum-1- e apum-2- não apresentam fenótipo alterado, sugerindo redundância funcional. Plantas com a expressão dessas proteínas afetadas por RNA antisense apresentaram folhas cloróticas e reduzidas, raízes mais curtas e menos ramificadas e baixa fertilidade, fenótipo semelhante ao de plantas que superexpressam KRP-2, um inibidor de CDK. O transcrito KRP-2 apresenta um elemento de ligação AraPum no 3\'-UTR sugerindo ser um possível alvo para APUM. Em adição, plantas antisense têm aumento de transcritos KRP-2 em relação a selvagens. Assim, foi proposto que essas proteínas agem coordenando a formação de folhas e raízes pela influência na tradução de KRP-2. A função ancestral das proteínas PUF de manter o ciclo celular em detrimento da diferenciação, parece ser conservada em plantas. / The PUF family is a group of conserved proteins that bind to rnRNAs regulating its stability and translation in key developrnental processes. Among the twenty five Arabidopsis proteins with PUF repeats, we found that three highly similar members, APUM-I, APUM-2 and APUM-3 (~90% identity) and co-localize spatially and temporally in the shoot apical and axillaries meristems, root elongation zone and pericycle during callus and lateral root formation, as well as stamens and pollens. RTPCR assays showed that these proteins have similar expression profiles in ali organs analyzed. Moreover, plant apum-1 and apum-2 knockouts have no detectably altered phenotype, suggesting a functional redundancy between them. Plants in which APUM-I, APUM-2 and APUM-3 expression were reduced through antisense RNA, showed chlorotic and reduced leaves, shorter and less ramificated roots and low fertility. This phenotype is similar to that of plants over-expressing the KRP-2 gene, a CDK inhibitor. An AraPum binding element at 3\'-UTR of the KRP-2 transcript suggests that it may be a possible target for APUM. In addition, in comparison to wild-type plants, antisense plants have increased KRP-2 transcripts levels. We proposed that APUM proteins act by coordinating leaf and root formation by way of influencing KRP-2 transiation. The ancestral function of PUF proteins in the maintenance of the cell cycle, to detriment of differentiation, seems to be conserved in plants.