Tesis sobre el tema "Floral meristem"

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.

Flowering is the most important process in a plant’s life as it is the essential step for its reproduction. Flower development starts with a tightly regulated process called the floral transition in which different regulatory pathways, which are regulated by environmental and internal signals, culminate in the transition from vegetative to reproductive growth. Subsequently, flowers develop instead of leaves and the formation of these flowers is controlled by complex regulatory pathways. In model organism Arabidopsis thaliana there are at least five different pathways that regulate the floral transition to guarantee that it occurs under the best possible conditions. The signals derived from these pathways are than integrated at the level of the floral pathway integrators which are LEAFY (LFY), FLOWERING LOCUS T (FT), SOPPRESSOR OF OVEREXPRESSION OF CO (SOC1). These genes are responsible for the switch from the shoot apical meristem (SAM) to the inflorescence meristem (IM) and are involved in the activation of the floral meristem identity (FMI) genes: LFY, LATE MERISTEM IDENTITY1 (LMI1), APETALA1 (AP1), CAULIFLOWER (CAL), SHORT VEGETATIVE PHASE (SVP) and AGAMOUS-LIKE 24 (AGL24). In fact, after the floral transition, the inflorescence meristem (IM) starts to produce floral meristems from its flanks. These meristems remain undifferentiated until stage 3 of flower development, thanks to the action of the FMI genes; afterwards, when some of these genes become repressed, the floral organs start to differentiate. In these first undifferentiated stages, the floral meristem grows and produces enough cells to support the subsequent differentiation of all the floral organs. Many of the genes involved in these two processes, floral transition and floral meristem determination, are MADS-box transcription factors. The MADS-box family is one of the best-characterized gene families in Arabidopsis and the its members represent key regulators of developmental processes. MADS-box factors are combinatorial proteins that act via multimerization and interact with other regulatory proteins in complexes to regulate the transcription of target genes. The aim of this thesis is the analysis of the genetic interactions of MADS-box transcription factors playing key roles during the floral transition and early stages of flower development. The floral pathways integrator SOC1 is a MADS-box gene that integrates at least four pathways that control flowering (photoperiod, vernalization, autonomous and gibberellin pathways), giving rise to the activation of the floral meristem identity genes (Parcy, 2005). In chapter 2, , we show that AGAMOUS-LIKE 42 (AGL42), AGAMOUS-LIKE 71 (AGL71) and AGAMOUS-LIKE 72 (AGL72) that are phylogenetically related to SOC1, are also involved in the floral transition of both the shoot apical meristem and axillary meristems and moreover, are involved in the gibberellin pathway. The soc1 agl42 ami::agl71-72 mutant shows an aerial rosettes bearing nodes phenotype. Our findings suggest that the SOC1-like genes are involved in the floral transition especially in the axillary meristem and the GA pathway is the main player controlling flowering in these axillary meristems both under short day and long day conditions. Furthermore SOC1 is able to directly control the expression of AGL42, AGL71 and AGL72 to maintain a proper expression level of SOC1-like genes. In chapter 3, the interactions between the floral meristem identity genes SVP, AGL24, AP1, CAL, which are all MADS-box transcription factors, and LFY is described. The lfy mutant shows partial reversions of flowers in inflorescence shoot-like structures and this phenotype is enhanced in the lfy ap1 double mutant. Here we show that combining the lfy mutant with agl24, svp single or agl4 svp double mutant enhances the lfy phenotype and that the agl24 svp lfy triple mutant phenocopies the ap1 lfy double mutant. Analysis of the molecular interactions between LFY and AGL24 and SVP showed that LFY is, together with AP1, a repressor of AGL24 and SVP whereas AGL24 and SVP positively regulate AP1 and LFY by direct binding to their regulatory regions. Since all genes are important to establish floral meristem identity this regulatory loop is probably important to maintain the correct relative expression levels of these genes. In chapter 4, we focalize our attention on SVP, a MADS-box gene involved in floral repression, before the floral transition, and in floral meristem (FM) identity determination, after the floral transition. An interesting feature of SVP is that it is the only Floral Meristem Identity Gene identified so far that is expressed exclusively in the undifferentiated FM. To date some transcription factors that are able to bind the SVP genomic region have already been identified by ChIP experiments, but it is still not clear how this gene is regulated. To understand this as a first step we are interested in the identification of the SVP minimal promoter region that fully comprises all its regulatory elements. We use, for our studies, lines that contain different SVP promoter fragments, that are cloned as transcriptional or translational fusions to the uidA gene, that encodes the beta-glucuronidase enzyme. This studies show that at least two regions are necessary for normal SVP expression: a 1 Kb fragment, located from 3000 to 2000 bp upstream of the start codon, and the first intron. In fact constructs lacking one of these two regions aren’t able to express GUS in the flower primordia. In conclusion this work contributes to get a better understanding of what exactly happens during the floral transition and, afterwards, in undifferentiated flower meristems.

[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
TESIS

Aerial growth of Arabidopsis thaliana is realized by the shoot apical meristem (SAM), which contains stem cells whose regular divisions sustain the continuous production of new organs. During reproductive development, the SAM produces flower meristems (FMs) on its flanks. Contrary to the SAM that generated them, FMs do not grow indefinitely and produce flowers, which are determinate structures, with a fixed number of organs. This determinacy is due to the repression of WUSHEL (WUS), which confers their identity to stem cells, by the homeotic gene AGAMOUS (AG). This arrest of stem cell maintenance is linked to the female developmental program of the flower, and requires SUPERMAN (SUP), which establish the boundary between the male an female parts of the flower. During my doctorate, I realized a functional analyses of three genes, REBELOTE (RBL), SQUINT (SQN) and ULTRAPETALA1 (ULT1). Combined mutation of two of these genes, or one of them and CRABS CLAW (CRC) triggers a strong loss of FM termination, with numerous supernumerary organs being produced. The range of phenotypes we got suggests that disruption of stem cell maintenance within the FM is a progressive process, which is redundantly controlled by several genes. Genetic and molecular analyses show that our mutant phenotypes result from a down regulation of AG in a sub-domain of its expression pattern, in the centre of FM. However, this decrease of AG expression is insufficient to explain all the phenotypes we observed, and genetic data suggest that RBL, SQN and ULT1 also influence SUP function. Additionally, preliminary analyses support a role for SQN in the CLAVATA pathway, while RBL may influence microRNAs biosynthesis. Finally RBL, SQN and ULT1 seem to contribute to flower homeostasis
La croissance aérienne d’Arabidopsis thaliana est assurée par le méristème apical caulinaire (MAC), qui contient des cellules souche dont les divisions permanentes permettent la mise en place continuelle de nouvelles structures. Au cours du développement reproducteur, le MAC produit des méristèmes floraux (MFs) sur ses flancs. Contrairement au MAC et bien qu’ils en soient issus, les MFs ne présentent pas de croissance indéfinie et produisent des fleurs, qui sont des structures déterminées, constituées d’un nombre fixe d’organes. Cette détermination est liée à la répression du gène WUSHEL (WUS), qui confère leur identité aux cellules souche, par le gène homéotique AGAMOUS (AG). Cet arrêt de l’entretien des cellules souche au sein du MF est lié à la mise en place des organes femelles de la fleur, les carpelles, et requiert l’action de SUPERMAN (SUP), qui permet l’établissement de la frontière entre les parties mâle et femelle de la fleur. Le travail de cette thèse consiste en la caractérisation de trois gènes, REBELOTE (RBL), SQUINT (SQN) et ULTRAPETALA1 (ULT1). La mutation combinée de 2 de ces gènes, ou de l’un d’entre eux et de CRABS CLAW (CRC), entraîne une perte marquée de l’arrêt du MF, qui continue alors indéfiniment à produire de nouveaux organes. La gamme de phénotypes obtenus suggère que l’arrêt de l’entretien des cellules souche au centre du MF est un phénomène progressif, contrôlé de manière redondante par plusieurs gènes. Une analyse génétique et moléculaire montre que les phénotypes obtenus résultent d’une baisse d’expression d’AG dans une partie interne de son domaine d’expression, au centre du MF. Cependant, ce défaut d’expression d’AG est insuffisant pour expliquer tous les phénotypes observés, et les données génétiques obtenues suggèrent que RBL, SQN et ULT1 influencent aussi l’activité de SUP. Enfin, des études préliminaires suggèrent que SQN pourrait influencer AG via la voie CLAVATA (CLV), tandis que RBL semble jouer sur la biosynthèse des microARNs, dont une famille, miR172, affecte l’activité d’AG. Finalement, RBL, SQN et ULT1 semblent contribuer à l’homéostasie du développement floral

Morphological, cell cycle and molecular events were studied during floral evocation in the shoot terminal meristem of the short-day plant Pharbitis nil. The haploid genome size of P.nil was 3.7 X 10e8 bp. Morphological and cell doubling time observations demonstrated that the apex widened and flattened follOwing an inductive dark treatment, the cell doubling time decreased in the peripheral zone and increased in the central zone. An inductive treatment comprising 48 h darkness given to 4 d old plants produced. 100~ flowering at the shoot terminal meristem. An inhibitory treatment was developed using 5 min RL breaks during the 48 h dark period. This treatment prevented flowering at the shoot terminal apex and discriminated between events essenttat for flowering and changes resulting from shifts from light to darkness and vice versa. It was shown using shading experiments that the shoot terminal apex may be directly involved in the reception of red-light. Maximal differences in the proportion of cells in G2/G 1 (G2/G 1 ratio) were recorded at the light/dark transition (i.e. at the end of the 48 h dark period). Apices were sampled at this time to construct floral and vegetative cDNA libraries. Less growth occurred in the central zone of the shoot apex. For example, the mitotic index of the central zone was consistently lower than that of the peripheral zone in both vegetative and induced apices. Unusual PLM curves were generated which meant that the component phases of the cell cycle were determined from microdensitometric data. The duration of the cell cycle in the peripheral zone was two-fold longer in vegetative apices compared to floral apices. However, the proportion of cycling cells (the growth fraction) was similar in the peripheral zones of both vegetative and floral apices. Following exposure to RL the duration of G2 in the peripheral zone increased five-fold. mRNA was separately extracted from 40 floral and vegetative shoot apices. The mRNA was copied into cDNA and amplified using the polymerase chain reaction. The amplified cDNA was then cloned into two A phage cDNA libraries (floral and vegetative). These libraries will form the foundation of future work on the molecular biology of floral evocation.

The LFY gene, which has now been isolated in at least 17 species, is important in the transition from vegetative to reproductive growth. The floral meristems of lfy mutants exhibited increased inflorescence characteristics, and constitutive expression of the gene was sufficient to convert lateral inflorescence meristems to solitary flowers in Arabidopsis, tobacco and Populus. Previous work on the determinate plant, Silene coeli-rosa, which required 7 LD for 100% flowering, concentrated on changes to the cell cycle and peptide composition of the shoot apex during floral evocation. A partial cDNA clone of a Silene LFY homologue (SFL) has been isolated. SFL shows strong homology to other LFY homologue proteins within the two conserved domains, with up to 88% identical amino acids. It contains highly acidic and basic domains, a glutamine rich region and leucine repeats: all putat5ive transcriptional activation domains. Expression studies using quantitative PCR show that SFL was not induced by non-inductive SD conditions, or by a continuous light treatment that inhibited flowering. This is in contrast to the expression patterns observed in vegetative Arabidopsis, pea, petunia, Impatiens, tobacco and tomato, but consistent with the expression of Antirrhinum homologue which is restricted to the floral meristems. During the 7 LD induction period, SFL transcripts were first detected after 5 LD, a treatment which resulted in 81% of plants flowering, under the conditions used. Fewer than 5 LD failed to induce flowering or SFL expression. SFL was also expressed in apices subjected to an inductive 7 LD treatment followed by 48h darkness, which delayed flowering and suppressed the synchronisation of the cell cycle which occurs immediately prior to floral initiation. In situ hybridisation revealed the spatial expression of SFL in Silene. No SFL was detected prior to D7 during floral induction or in non-inductive SD controls. On experimental D7, SFL mRNA was restricted to the flanks of the primary apical dome and in D8 apices expression had spread throughout the dome. Importantly, this pattern of expression differs to that observed in the other two determinate species in which LFY has been studied, namely tobacco and Impatiens.

Les plantes ont la capacité à former de nouveaux organes grâce à une croissance continue assurée par une réserve de cellules souches au sein de structures spécifiques, les méristèmes. Les méristèmes floraux diffèrent des méristèmes végétatifs par leur caractère déterminé aboutissant à la production des fleurs. Le gène IMA (INHIBITOR OF MERISTEM ACTIVITY) code une protéine contenant un motif «doigt à zinc» (MIF) régulant les processus développementaux de la fleur et des ovules chez la tomate. En effet, IMA inhibe la prolifération cellulaire au cours de la terminaison florale en agissant sur l’expression du gène WUSCHEL, responsable du maintien du pool de cellules souches et contrôle le nombre de carpelles (Sicard et al., 2008). De plus, les protéines IMA et son orthologue chez Arabidopsis, MIF2, modulent la réponse à certaines phytohormones. De manière identique à la protéine MIF1 (Hu and Ma, 2006), IMA/MIF2 régule négativement la réponse aux brassinostéroïdes, à l’auxine, aux cytokinines et aux gibbérellines mais positivement la réponse à l’acide abscissique suggérant une fonction commune des protéines MIF dans les voies de réponse aux phytohormones. Un criblage d’une banque d’ADNc par la technique de double hybride a permis de révéler l’interaction entre les protéines IMA/MIF2 et une sous-unité du complexe signalosome, CSN5. De façon intéressante, les plantes mutantes csn5 d’Arabidopsis montrent de nombreuses altérations phénotypiques telles qu’un aspect buissonnant résultant de la perte de la dominance apicale, et une altération de la réponse à l’obscurité et à l’auxine. Ces phénotypes sont fortement ressemblants aux phénotypes des plantes MIF1OE d’Arabidopsis (Hu and Ma, 2006) et des plantes IMAOE de tomate (Sicard et al., 2008). Les résultats obtenus au cours de ce projet montrent que la protéine IMA inhibe la fonction du complexe signalosome grâce à son interaction avec la protéine CSN5
Plants have the ability to form new organs as a result of indeterminate growth ensured by specific regions of pluripotent cells, called meristems. Flowers are produced by the activity of floral meristems which differ from vegetative meristems in their determinate fate. The INHIBITOR OF MERISTEM ACTIVITY (IMA) gene encoding a Mini Zinc Finger (MIF) protein from tomato (Solanum lycopersicum) regulates the processes of flower and ovule development. IMA inhibits cell proliferation during floral termination, controls the number of carpels during floral development and acts as a repressor of the meristem organizing centre gene WUSCHEL (Sicard et al., 2008). We demonstrated that IMA and its Arabidopsis ortholog MIF2 is also involved in a multiple hormonal signalling pathway, as a putative conserved feature for plant MIF proteins (Hu and Ma, 2006). Alike Arabidopsis MIF1, IMA/MIF2 regulates negatively BR, auxin, cytokinin and gibberellin signalling and positively ABA signaling. Using yeast two-hybrid screening experiments, we identified a strong protein-protein interaction between IMA and the signalosome subunit 5 (CSN5). Interestingly the csn5 mutant in Arabidopsis displays pleiotropic developmental defects such as a bushy phenotype originating from the loss of apical dominance and the alteration in sensitivity to darkness and auxin signals. These phenotypes are strikingly similar to what was described for Arabidopsis MIF1 (Hu and Ma, 2006) and tomato IMA overexpressors plants (Sicard et al., 2008), respectively. Taken together our data strongly suggest that IMA may act as an inhibitor of CSN function through its physical interaction with SlCSN5. The observed converse effects of IMA/MIF2 overexpression or deregulation on plant development and the abundance of developmental marker genes further support the notion of a CSN inhibitory control, since the COP9 signalosome through the specific deneddylation activity of the CSN5 subunit regulates plant hormone signalling

Thesis (Ph. D.)--University of California, Riverside, 2010.
Includes abstract. Title from first page of PDF file (viewed May 18, 2010). Includes bibliographical references. Issued in print and online. Available via ProQuest Digital Dissertations.

O abacateiro (Persea americana Mill.) possui desenvolvimento organizado em fluxos de crescimento e florescimento em panículas, provenientes principalmente de gemas terminais, podendo ocorrer em menor intensidade a partir de gemas axilares. Dessa forma, o presente estudo teve como objetivo avaliar a capacidade de florescimento e a presença de substâncias ergásticas nas células de gemas axilares, bem como determinar a contribuição dos fluxos de crescimento de primavera e verão para a composição floral dos abacateiros \'Geada\', \'Fortuna\', Quintal\', \'Margarida\' e \'Hass\', localizados no sudoeste do Estado de São Paulo, Brasil. Adotou-se o delineamento experimental inteiramente casualizado e por meio da contagem do número de brotações e inflorescências, o acompanhamento do desenvolvimento vegetativo e reprodutivo foi feito em 40 ramos do fluxo de primavera e 40 ramos do fluxo de verão, distribuídos em cinco plantas por cultivar, no período de março a agosto/2016. A capacidade de florescimento de gemas axilares foi avaliada em estruturas coletadas mensalmente entre março e julho de 2016 nos ramos dos fluxos de primavera e verão, sendo as alterações anatômicas do meristema e a presença de substâncias ergásticas (amido, proteínas totais, compostos fenólicos e polissacarídeos) monitoradas a partir de testes histológicos e histoquímicos. Para as análises histológicas, as amostras vegetais foram desidratadas em série gradual de álcoois, emblocados em historesina e coradas em coloração dupla com reagente ácido periódico de Schiff e Naftol Blue Black. O florescimento de todas as cultivares ocorreu no mês de agosto/2016 e a formação de inflorescências foi predominante em ramos provenientes do fluxo de verão para todas as cultivares. Os resultados evidenciam a capacidade de florescimento de gemas axilares dos abacateiros, as quais são anatomicamente idênticas às gemas terminais e apresentaram início do comprometimento com o florescimento, caracterizada pelo aparecimento dos eixos secundários da inflorescência, dois meses antes (entre maio e julho) da época de floração (agosto/setembro).
Avocado trees (Persea americana Mill.) has an organized development in fluxes of growth and flowering in panicles, mainly coming from terminal buds, and may occur in less intensity from axillary buds. Thus, the present study had as objective the evaluation of the flowering potential and presence of ergastic substances in axillary buds, as well as to determine the contribution of the spring and summer fluxes growth to the floral composition of the avocado trees \'Geada\', \'Fortuna\' , Quintal \',\' Margarida \'and\' Hass\', located in the southwest of São Paulo State, Brazil. The experimental design was completely randomized and the vegetative and reproductive development was monitored in 40 branches of the spring flux and 40 branches of the summer flux by counting the number of shoots and inflorescences, distributed in five plants per cultivar, in the period from March to August / 2016. The flowering capacity of axillary buds was evaluated in monthly collected structures between March and July of 2016 in the branches of spring and summer fluxes, being the anatomical alterations of the meristem and the presence of ergastic substances (starch, total proteins, phenolic compounds and polysaccharides) monitored from histological and histochemical analyses. For the histological analyzes, the plant samples were dehydrated in a gradual series of alcohols, placed in historesin and stained in double staining with periodic acid reagents of Schiff and Naftol Blue Black. The flowering of all cultivars occurred in August / 2016 and inflorescence formation was more significant in branches from the summer flow for all cultivars. The results showed the flowering ability of axillary buds of the avocado trees, which are anatomically identical to the terminal buds and showed the beginning of the flowering, characterized by the appearance of the secondary axes of the inflorescence two months before (between May and July) of flowering (August / September).

Setaria viridis é uma espécie de gramínea muito relevante em estudos de desenvolvimento vegetal, por determinadas características que a fazem uma excelente proposta de organismo modelo para plantas monocotiledôneas de metabolismo C4. Para que a espécie seja utilizada amplamente em estudos que visem compreender o funcionamento e desenvolvimento vegetal, bem como os mecanismos moleculares que o modulam, é essencial que aspectos de seu desenvolvimento sejam desvendados. Nesse contexto, a caracterização do meristema radicular e das estruturas presentes nos estágios iniciais da germinação é importante para se compreender como ocorre, em gramíneas, o surgimento de tipos de raízes diferentes, além de correlacionar quais fatores ambientais e endógenos estão envolvidos na escolha de diferentes arquiteturas de sistemas radiculares em gramíneas. Além disso,a caracterização do desenvolvimento de estruturas florais em Setaria viridis é importante, uma vez que traz informações que podem contribuir com um aumento na eficiência de metodologias de transformação genética para a espécie, via \"spike dip\". O presente trabalho caracterizou morfoanatomicamente o desenvolvimento radicular desde os estágios iniciais da germinação e o desenvolvimento floral em Setaria viridis. Além disso, buscou estabelecer relação entre o surgimento de diferentes tipos de raízes (primárias e as adventícias) com diferentes condições de luminosidade em cultivos in vitro
Setaria viridis is a very important grass in studies of plant development, due to some characteristics that make it an excellent proposed model organism for monocotyledonous plants with C4 metabolism. To be widely used in studies that aim to understand plant functioning and development, as well as the molecular mechanisms that modulate it, it is essential that aspects of its development be unraveled. In this context, the characterization of the root meristem and structures present in the early stages of germination is also important to understand how the emergence of different root types occurs in grasses. Moreover, to correlate the environmental and endogenous factors involved in the choice of different architectures of root systems in grasses. Furthermore, the characterization of the development of floral structures in Setaria viridis is important, since it brings information that can contribute to the efficiency of methodologies of genetic transformation for the species. The present work characterized morphologically the root development during the initial stages of germination and the floral development of Setaria. In addition, it sought to establish a relationship between the emergences of different types of roots (primary and adventitious) with different light conditions in in vitro cultures

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Eriocaulaceae é uma família de monocotiledôneas bastante representativa na flora brasileira. Estudos sobre a sua morfologia, anatomia e desenvolvimento florais têm tido um papel importante na resolução de questões taxonômicas e evolutivas. Paepalanthus, seu maior gênero, apresenta morfologia bastante complexa e suas relações infragenéricas ainda não são totalmente conhecidas. Neste trabalho são estudadas a anatomia e o desenvolvimento floral de oito espécies dímeras de Paepalanthus pertencentes aos diferentes clados em que essa característica ocorre, buscando maior conhecimento do gênero e das relações entre as espécies dos clados estudados: P. subg. Thelxinoë, P. sect. Conodiscus, P. sect. Diphyomene, P. sect. Eriocaulopsis e P. ser. Dimeri. O presente trabalho contribui ao encontrar caracteres anatômicos e ontogenéticos florais com valor taxonômico e que auxiliam na elucidação de relações evolutivas em Paepalanthus. Na flor estaminada, o verticilo externo de estaminódios, previamente relatado por diferentes autores, não se desenvolve e é corretamente descrito como a região apical das pétalas. As flores pistiladas desenvolvem ovário com uma porção sinascidiada proximal e uma pequena porção simplicada apical, descritas pela primeira vez em Eriocaulaceae. Também são descritos três padrões para o gineceu das espécies estudadas de Paepalanthus dímeros: a) gineceu com dois ramos nectaríferos e dois ramos estigmáticos livres (P. sect. Diphyomene, P. sect. Eriocaulopsis e P. ser. Dimeri); b) gineceu com dois ramos nectaríferos, mas com os ramos estigmáticos fusionados na flor madura até a altura dos estigmas (P. sect. Conodiscus); e c) gineceu em que os ramos nectaríferos não se desenvolvem e os ramos estigmáticos são fusionados (P. subg. Thelxinoë). A ocorrência de ramos nectaríferos e ramos estigmáticos livres pode ser uma sinapomorfia para o clado que compreende P. sect. Diphyomene, P. sect. Eriocaulopsis e P. ser. Dimeri. A presença de ramos estigmáticos fusionados corrobora a proximidade P. sect. Conodiscus e P. subg. Thelxinoë. Já a ausência de ramos nectaríferos pode ser considerada uma sinapomorfia de P. subg. Thelxinoë. Devido à homologia entre os pistilódios das flores estaminadas e os ramos nectaríferos das flores pistiladas, o desenvolvimento e a anatomia dessas estruturas são semelhantes e algumas características morfológicas e do revestimento se mostram diagnósticas para a diferenciação das espécies. Por sua vez, os tricomas encontrados nas peças florais estéreis das espécies estudadas também diferem entre as categorias infragenéricas e são importantes para distingui-las.
Eriocaulaceae is a well-represented monocot family in the Brazilian flora. Studies about its floral morphology, anatomy and development have played an important role in solving taxonomic and evolutionary problems within the family. Paepalanthus, the larger genus in Eriocaulaceae, has a very complex morphology, and its infrageneric relationships are not completely known so far. In this work, we studied the floral anatomy and development of eight species of Paepalanthus belonging to distinct clades in which the dimery occurs, in order to improve our knowledge about the genus and the relationships between the species of the studied clades: P. subg. Thelxinoë, P. sect. Conodiscus, P. sect. Diphyomene, P. sect. Eriocaulopsis, and P. ser. Dimeri. The present work contributes by finding floral anatomical and ontogenetical characters with taxonomic significance and also characters that help clarify the relationships in Paepalanthus. In the staminate flower, the outer whorl of staminodes, previously reported by different authors, does not grow and it is correctly described as the apical portion of petals. Pistillate flowers develop ovary with a proximal synascidiate portion and a short apical symplicate portion, reported for the first time in Eriocaulaceae. Furthermore, three patterns of gymnoecium are described for the studied species of dimerous Paepalanthus: a) gymnoecium with two nectariferous branches and two free stigmatic branches (P. sect. Diphyomene, P. sect. Eriocaulopsis e P. ser. Dimeri); b) gymnoecium with two nectariferous branches, and stigmatic branches fused up to the stigma level (P. sect. Conodiscus); and c) gymnoecium in which the nectariferous branches do not develop and the stigmatic branches are fused (P. subg. Thelxinoë). The occurrence of nectariferous branches and free stigmatic branches may be a synapomorphy of the clade that includes P. sect. Diphyomene, P. sect. Eriocaulopsis e P. ser. Dimeri. The occurrence of fused stigmatic branches supports the proximity between P. sect. Conodiscus e P. subg. Thelxinoë. Yet, the absence of nectariferous branches may be considered a synapomorphy of P. subg. Thelxinoë. Due to the homology between nectariferous pistillodes found in staminate flowers and nectariferous branches found in pistillate flowers, the development and anatomy of both structures are similar, and some morphological and epidermal features showed diagnostic for the distinction of the species. Moreover, trichomes were found in sterile floral structures of the studied species. They also differ among the infrageneric categories and are important to distinguish them.

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

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

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.

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.

LFY of Arabidopsis is a member of a unique plant specific transcription factor family. It is involved in giving meristem a determinate floral fate by the activation of floral organ identity genes and preventing inflorescence meristem identity. RFL is a homolog of FLO/LFY in rice. Studies from our lab on rice RFL, based on the effects of knockdown or overexpression, showed its major functions are in timing the conversion of SAM to IM and to prevent the premature conversion of branch meristem to spikelets. Additionally roles in vegetative axillary meristem specification have been also been identified in laboratory. Here, we attempt to delineate molecular pathways directly regulated by RFL as a transcription factor controlling inflorescence and floral development in rice. Part I: Identification of global target genes bound by RFL in developing rice inflorescences We carried out ChIP sequencing of the DNA bound by RFL in panicles (01.-0.3cm stage) using anti-RFL antibody. DNA sequences in one library pool were analyses by the MACS algorithm (FDR<0.01), to find 8000 binding sites while the SPP algorithm identified 5000 enriched peaks. These mapped to 2500 or 2800 gene-associated loci respectively, 617 of which were common loci to both pipelines. Several RFL bound gene loci were homologs of Arabidopsis thaliana LFY gene targets. Such gene targets underscore conserved downstream targets for LFY-proteins in evolutionarily very distinct species. AtLFY is known to bind variants of CCANT/G cis element classified as primary, inflorescence or seedling type. We scanned for these three types of cis elements at 123 RFL bound genes with likely functions in flowering. For a few of these 123 rice loci we find one of these cis motifs (p-value<0.001) in RFL bound ChIP-seq data. To validate these targets of RFL, we adopted in vitro DNA-protein binding assays with bacterially purified RFL protein. We confirm RFL target interactions with some genes implicated in flowering time, others in photoperiod triggered flowering, circadian rhythm, gibberellin hormone pathway, inflorescence development and branching. The in vitro experiments hint different RFL-DNA binding properties as compared to Arabidopsis LFY. We report binding to sequences at rice gene loci that are unique targets. Part II: Pathways regulated by RFL for reproductive transition and panicle development To co-relate DNA binding of RFL to target loci with changes in their gene expression, expression studies were taken up for selected set of genes implicated in rice flowering transition and panicle architecture. To study in planta and tissue specific gene regulation by RFL we raised RFL dsRNAi transgenics. Comparative transcript analysis in these RFL partial knockdown lines and matched wild type tissues reveal that RFL is an activator for some genes and repressor for other gene targets. We also examined if the gene expression effects of RFL knockdown can be reversed by induced complementation with an RFL-GR protein. We raised transgenics plants with a T-DNA ubi:RFL-GR, 35S CaMV:amiR RFL for these experiments. In planta target gene transcript levels were assessed in various conditions conditions. These studies validate rice RFL as an activator of some panicle architecture genes. Part III: Analysis of endogenous RFL protein in WT rice tissues Studies in Arabidopsis and in petunia with LFY and AFL, respectively, implicate these some abnormal mobility as compared to their predicted molecular weight when overexpressed. We studied endogenous RFL protein abundance in planta, adopting western analysis with anti-RFL antibody. We consistently identify two prominent cross reacting bands in different tissues which can be also be pulled-down from whole nuclear extracts of panicle and axillary meristem tissues. We speculate on likely modifications and possible functions for the same.

LFY of Arabidopsis is a member of a unique plant specific transcription factor family. It is involved in giving meristem a determinate floral fate by the activation of floral organ identity genes and preventing inflorescence meristem identity. RFL is a homolog of FLO/LFY in rice. Studies from our lab on rice RFL, based on the effects of knockdown or overexpression, showed its major functions are in timing the conversion of SAM to IM and to prevent the premature conversion of branch meristem to spikelets. Additionally roles in vegetative axillary meristem specification have been also been identified in laboratory. Here, we attempt to delineate molecular pathways directly regulated by RFL as a transcription factor controlling inflorescence and floral development in rice. Part I: Identification of global target genes bound by RFL in developing rice inflorescences We carried out ChIP sequencing of the DNA bound by RFL in panicles (01.-0.3cm stage) using anti-RFL antibody. DNA sequences in one library pool were analyses by the MACS algorithm (FDR<0.01), to find 8000 binding sites while the SPP algorithm identified 5000 enriched peaks. These mapped to 2500 or 2800 gene-associated loci respectively, 617 of which were common loci to both pipelines. Several RFL bound gene loci were homologs of Arabidopsis thaliana LFY gene targets. Such gene targets underscore conserved downstream targets for LFY-proteins in evolutionarily very distinct species. AtLFY is known to bind variants of CCANT/G cis element classified as primary, inflorescence or seedling type. We scanned for these three types of cis elements at 123 RFL bound genes with likely functions in flowering. For a few of these 123 rice loci we find one of these cis motifs (p-value<0.001) in RFL bound ChIP-seq data. To validate these targets of RFL, we adopted in vitro DNA-protein binding assays with bacterially purified RFL protein. We confirm RFL target interactions with some genes implicated in flowering time, others in photoperiod triggered flowering, circadian rhythm, gibberellin hormone pathway, inflorescence development and branching. The in vitro experiments hint different RFL-DNA binding properties as compared to Arabidopsis LFY. We report binding to sequences at rice gene loci that are unique targets. Part II: Pathways regulated by RFL for reproductive transition and panicle development To co-relate DNA binding of RFL to target loci with changes in their gene expression, expression studies were taken up for selected set of genes implicated in rice flowering transition and panicle architecture. To study in planta and tissue specific gene regulation by RFL we raised RFL dsRNAi transgenics. Comparative transcript analysis in these RFL partial knockdown lines and matched wild type tissues reveal that RFL is an activator for some genes and repressor for other gene targets. We also examined if the gene expression effects of RFL knockdown can be reversed by induced complementation with an RFL-GR protein. We raised transgenics plants with a T-DNA ubi:RFL-GR, 35S CaMV:amiR RFL for these experiments. In planta target gene transcript levels were assessed in various conditions conditions. These studies validate rice RFL as an activator of some panicle architecture genes. Part III: Analysis of endogenous RFL protein in WT rice tissues Studies in Arabidopsis and in petunia with LFY and AFL, respectively, implicate these some abnormal mobility as compared to their predicted molecular weight when overexpressed. We studied endogenous RFL protein abundance in planta, adopting western analysis with anti-RFL antibody. We consistently identify two prominent cross reacting bands in different tissues which can be also be pulled-down from whole nuclear extracts of panicle and axillary meristem tissues. We speculate on likely modifications and possible functions for the same.

The main objective of this research project was to study the growth and development of the floral nectar spur of Centranthus ruber (L.) DC. Nectar spurs are tubular floral outgrowths, generally derived from the perianth organs, which typically contain secreted floral nectar. The morphological characteristics of the spur, particularly the length, determine which floral visitors will be able to access the nectar reward pooled at the spur tip. Therefore, nectar spurs are ecologically important for the development of specialised pollinator interactions and have been demonstrated to act as key innovations in the evolution of some taxa. Morphological and anatomical characteristics of the spur and floral nectary were investigated using light and scanning electron microscopy. Ultrastructural features of the nectar spur, particularly the floral nectary within, were assessed using transmission electron microscopy. Nectar in C. ruber is produced by a trichomatous nectary which runs along the entire, inner abaxial surface of the spur. The nectary is aligned with the single vascular bundle which runs along the abaxial side of the spur, through the sub-nectary parenchyma, and back up the adaxial side. The secretory trichomes are unicellular and, in late development, they develop a thick layer of secondary wall ingrowths which vastly increases the surface area of the plasma membrane for nectar secretion. Elongate, non-secretory trichomes occupy the entire remaining circumference of the spur’s inner epidermis, but their density is reduced compared to the secretory trichomes. The cellular basis for spur growth is poorly characterized in the literature. Until recently, it was assumed that all nectar spurs grow by the constant production of new cells via up to three potential meristematic regions (the meristem hypothesis, Tepfer 1953). The cellular basis for spur growth in C. ruber was investigated by cell file counts and cell length and width measurements along the lateral side of nectar spurs in each of the developmental stages. DAPI stained spurs were also examined with Confocal/Apotome microscopy to determine the timing and position of cell division activity throughout spur development. It was determined that elongation of the spur epidermal cells contributes much more to spur growth than cell division. In early development, division is the primary driver of spur growth and the cells are isotropic. However, as development progresses, cell division activity slows down and the spur cells become increasingly anisotropic until anthesis. The patterns of nectar secretion were determined by assessing the volume, solute concentration and carbohydrate composition of the nectar throughout flowering phenology in two C. ruber plants. Nectar volumes and solute amounts rose initially, followed by an eventual decline in both as phenology progressed towards senescence. Because this study was conducted on greenhouse grown plants, it can be assumed that nectar was not removed by insects, suggesting that it is likely reabsorbed following secretion. High performance liquid chromatography (HPLC) analysis determined that C. ruber's nectar is sucrose dominant and that nectar composition remains stable following anthesis throughout floral phenology.

In angiosperms, specialized reproductive structures are borne in flowers to ensure their reproductive success. After the vegetative growth, plants undergo reproductive phase change to produce flowers. Floral meristems (FMs) are generated on the flanks of inflorescence and groups of specialized stem cells in the FM differentiate into four whorls of organs of a flower. In dicots, floral meristem successively gives rise to sepals, petals, stamens and carpels; after which it terminates. The fate of organs formed on FM is under the control of genetic regulators, key among which are members of MADS box transcription factor family. Their individual and combined act confers distinct identities to floral organs. Grass flowers are highly modified in structure. Rice flower, a model for grasses, is borne on a short branch called spikelet and they together from the basic structural units of the rice infloresences known as panicle. The outer whorl organs of a grass floret are bract-like structures known as lemma and palea to dicot sepals is highly dibated (see Chapter 1). In grass florets, petal homologs are a pair of highly reduced, fleshy bracts known as lodicules, while stamen and carpel homologs occupy the same position and share the same functions as their dicot counterparts. Aside from these distinct outer whorl organs, the florets are subtended by two pairs of bracts known as empty glumes and rudimentary glumes. The genetic regulators that control their unique identities and those that perform conserved functions are very intriguing and central questions in plant developmental biology. Using various contemporary and complementary technologies, we have analysed the molecular functions and downstream pathways of a MADS box transcription factor, OsMADSI during the rice floret meristem specification and organ development. Further by reverse genetics and overexpression studies, we have also functionally characterized two target genes of OsMADSI, OsETTINI and OsETTINI2 to understand their roles downstream to OsMADSI during the rice floret development.

In angiosperms, specialized reproductive structures are borne in flowers to ensure their reproductive success. After the vegetative growth, plants undergo reproductive phase change to produce flowers. Floral meristems (FMs) are generated on the flanks of inflorescence and groups of specialized stem cells in the FM differentiate into four whorls of organs of a flower. In dicots, floral meristem successively gives rise to sepals, petals, stamens and carpels; after which it terminates. The fate of organs formed on FM is under the control of genetic regulators, key among which are members of MADS box transcription factor family. Their individual and combined act confers distinct identities to floral organs. Grass flowers are highly modified in structure. Rice flower, a model for grasses, is borne on a short branch called spikelet and they together from the basic structural units of the rice infloresences known as panicle. The outer whorl organs of a grass floret are bract-like structures known as lemma and palea to dicot sepals is highly dibated (see Chapter 1). In grass florets, petal homologs are a pair of highly reduced, fleshy bracts known as lodicules, while stamen and carpel homologs occupy the same position and share the same functions as their dicot counterparts. Aside from these distinct outer whorl organs, the florets are subtended by two pairs of bracts known as empty glumes and rudimentary glumes. The genetic regulators that control their unique identities and those that perform conserved functions are very intriguing and central questions in plant developmental biology. Using various contemporary and complementary technologies, we have analysed the molecular functions and downstream pathways of a MADS box transcription factor, OsMADSI during the rice floret meristem specification and organ development. Further by reverse genetics and overexpression studies, we have also functionally characterized two target genes of OsMADSI, OsETTINI and OsETTINI2 to understand their roles downstream to OsMADSI during the rice floret development.

Plant reproductive development begins when vegetative shoot apical meristems change their fate to inflorescence meristems which develop floral meristems on the flanks. This process of meristem fate change and organ development involves regulated activation and/or repression of many cell fate determining factors that execute down-stream gene expression cascades. Flowers are formed when floral organs are specified on the floral meristem in four concentric whorls. In the model dicot plant Arabidopsis, the identity and pattern of floral organs is determined by combined actions of MADS-domain containing transcription factors of the classes A, B, C, D and E. Rice florets are produced on a compact higher order branch of the inflorescence and have morphologically distinct non-reproductive organs that are positioned peripheral to the male and female reproductive organs. These unique outer organs are the lemma and palea that create a closed floret internal to which are a pair of lodicules that are asymmetrically positioned fleshy and reduced petal-like organs. The unique morphology of these rice floret organs pose intriguing questions on how evolutionary conserved floral meristem specifying and organ fate determining factors bring about their distinct developmental functions in rice. We have studied the functions for two rice MADS-box proteins, OsMADS2 and OsMADS1, to understand their role as master regulators of gene expression during rice floret meristem specification and organ development. OsMADS2; a transcriptional regulator of genes expression required for lodicule development Arabidopsis B-function genes AP3 and PI are stably expressed in the whorl 2 and 3 organ primordia and they together with other MADS-factors (Class A+E or C+E) regulate the differentiation of petals and stamens (Jack et al, 1992; Goto and Meyerowitz, 1994). Rice has a single AP3 ortholog, SPW1 (OsMADS16) but has duplicated PI-like genes, OsMADS2 and OsMADS4. Prior studies in our lab on one of these rice PI-like genes OsMADS2 showed that it is needed for lodicule development but is dispensable for stamen specification (Kang et al., 1998; Prasad and Vijayraghavan, 2003). Functional divergence between OsMADS2 and OsMADS4 may arise from protein divergence or from differences in their expression patterns within lodicule and stamen whorls. In this study, we have examined the dynamic expression pattern of both rice PI-like genes and have examined the likelihood of their functional redundancy for lodicule development. We show OsMADS2 transcripts occur at high levels in developing lodicules and transcripts are at reduced levels in stamens. In fully differentiated lodicules, OsMADS2 transcripts are more abundant in the distal and peripheral regions of lodicules, which are the tissues that are severely affected in OsMADS2 knock-down florets (Prasad and Vijayraghavan, 2003). The onset of OsMADS4 expression is in very young floret meristems before organ primordia emergence and this is expressed before OsMADS2. In florets undergoing organogenesis, high level OsMADS4 expression occurs in stamens and carpels and transcripts are at low level in lodicules (Yadav, Prasad and Vijayraghvan, 2007). Thus, we show that these paralogous genes differ in the onset of their activation and their stable transcript distribution within lodicules and stamens that are the conserved expression domains for PI-like genes. Since the expression of OsMADS4 in OsMADS2 knock-down florets is normal, our results show OsMADS2 has unique functions in lodicule development. Thus our data show subfunctionalization of these paralogous rice PI-like genes. To identify target genes regulated by OsMADS2 that could contribute to lodicule differentiation, we have adopted whole genome transcript analysis of wild-type and dsRNAiOsMADS2 panicles with developing florets. This analysis has identified potential down-stream targets of OsMADS2 many of which encode transcription factors, components of cell division cycle and signalling factors whose activities likely control lodicule differentiation. The expression levels of few candidate targets of OsMADS2 were examined in various floret organs. Further, the spatial expression pattern for four of these down-stream targets of OsMADS2 was analysed and we find overlap with OsMADS2 expression domains (Yadav, Prasad and Vijayraghvan, 2007). The predicted functions of these OsMADS2 target genes can explain the regulation of growth and unique vascular differentiation of this short fleshy modified petal analog. OsMADS1, a rice E-class gene, is a master regulator of other transcription factors and auxin and cytokinin signalling pathways In Arabidopsis four redundant SEPALLATA factors (E-class) are co-activators of other floral organ fate determining MADS-domain factors (classes ABCD) and thus contribute to floral meristem and floral organ development (Krizek and Fletcher, 2005). Among the grass-specific sub-clade of SEP-like genes, rice OsMADS1 is the best characterized. Prior studies in our lab showed that OsMADS1 is expressed early throughout the floret meristem before organ primordia emergence and later is restricted to the developing lemma and palea primordia with weak expression in carpel (Prasad et al, 2001). Stable expression continues in these floret organs. OsMADS1 plays critical non-redundant functions to specify a determinate floret meristem and also regulates floret organ identities (Jeon et al., 2000; Prasad et al, 2001; 2005; Agarwal et al., 2005; Chen et al., 2006). In the present study, we have adopted two different functional genomic approaches to identify genes down-stream of OsMADS1 in order to understand its mechanism of action during floret development. We have studied global transcript profiles in WT and dsRNAiOsMADS1 panicles and find OsMADS1 is a master regulator of a significant fraction of the genome’s transcription factors and also a number of genes involved in hormone-dependent cell signalling. We have validated few representative genes for transcription factors as targets regulated by OsMADS1. In a complementary approach, we have determined the consequences of induced-ectopic over-expression of a OsMADS1:ΔGR fusion protein in shoot apical meristems of transgenic plants. Transcript levels for candidate target genes were assessed in induced tissues and compared to mock-treated meristems and also with meristems induced for OsMADS1:ΔGR but blocked for new protein synthesis. These analyses show that OsMADS55 expression is directly regulated by OsMADS1. Importantly, OsMADS55 is related to SVP that plays an important role in floral transition and floral meristem identity in Arabidopsis. OsHB3 and OsHB4, homeodomain transcription factors, with a probable role in meristem function, are also directly regulated by OsMADS1. The regulation of such genes by OsMADS1 can explain its role in floret meristem specification. In addition to regulating other transcription factors, OsMADS1 knock-down affects expression of genes encoding proteins in various steps of auxin and cytokinin signalling pathways. Our differential expression profiling showed OsMADS1 positively regulates the auxin signalling pathway and negatively regulates cytokinin mediated signalling events. Through our induced ectopic expression studies of OsMADS1:ΔGR, we show OsMADS1 directly regulates the expression of OsETTIN2, an auxin response transcription factor, during floret development. Overall, we demonstrate that OsMADS1 modulates hormonal pathways to execute its functions during floret development on the spikelet meristems. Functional studies of OsMGH3; an auxin-responsive indirect target of OsMADS1 To better understand the contribution of auxin signalling during floret development, we have functionally characterized OsMGH3, a down-stream indirect target of OsMADS1, which is a member of the auxin-responsive GH3 family. The members of this family are direct targets of auxin response factors (ARF) class of transcription factors. GH3-proteins inactivate cellular auxin by conjugating them with amino acids and thus regulate auxin homeostasis in Arabidopsis (Staswick et al., 2005). OsMGH3 expression in rice florets overlaps with that of OsMADS1 (Prasad et al, 2005). In this study, we have demonstrated the consequences of OsMGH3 over-expression and knock-down. The over-expression of OsMGH3 during vegetative development causes auxin-deficient phenotypes such as dwarfism and loss of apical dominance. Its over-expression in developing panicles that was obtained by driving its expression from tissue-specific promoters created short panicles with reduced branching. The latter is a phenotype similar to that observed upon over-expression of OsMADS1. In contrast, the down-regulation of endogenous OsMGH3 through RNA-interference produced auxin over-production phenotypes such as ectopic rooting from aerial nodes. Knock-down of OsMGH3 expression in florets affected carpel development and pollen viability both of which affect floret fertility. Taken together, this study provides evidence for the importance of auxin homeostasis and its transcriptional regulation during rice panicle branching and floret organ development. Our analysis of various conserved transcription factors during rice floret development suggest that factors like OsMADS2, OsMADS4 and OsMADS1 are master regulators of gene expression during floret meristem specification and organ development. The target genes regulated by these factors contribute to development of morphologically distinct rice florets.

Plant reproductive development begins when vegetative shoot apical meristems change their fate to inflorescence meristems which develop floral meristems on the flanks. This process of meristem fate change and organ development involves regulated activation and/or repression of many cell fate determining factors that execute down-stream gene expression cascades. Flowers are formed when floral organs are specified on the floral meristem in four concentric whorls. In the model dicot plant Arabidopsis, the identity and pattern of floral organs is determined by combined actions of MADS-domain containing transcription factors of the classes A, B, C, D and E. Rice florets are produced on a compact higher order branch of the inflorescence and have morphologically distinct non-reproductive organs that are positioned peripheral to the male and female reproductive organs. These unique outer organs are the lemma and palea that create a closed floret internal to which are a pair of lodicules that are asymmetrically positioned fleshy and reduced petal-like organs. The unique morphology of these rice floret organs pose intriguing questions on how evolutionary conserved floral meristem specifying and organ fate determining factors bring about their distinct developmental functions in rice. We have studied the functions for two rice MADS-box proteins, OsMADS2 and OsMADS1, to understand their role as master regulators of gene expression during rice floret meristem specification and organ development. OsMADS2; a transcriptional regulator of genes expression required for lodicule development Arabidopsis B-function genes AP3 and PI are stably expressed in the whorl 2 and 3 organ primordia and they together with other MADS-factors (Class A+E or C+E) regulate the differentiation of petals and stamens (Jack et al, 1992; Goto and Meyerowitz, 1994). Rice has a single AP3 ortholog, SPW1 (OsMADS16) but has duplicated PI-like genes, OsMADS2 and OsMADS4. Prior studies in our lab on one of these rice PI-like genes OsMADS2 showed that it is needed for lodicule development but is dispensable for stamen specification (Kang et al., 1998; Prasad and Vijayraghavan, 2003). Functional divergence between OsMADS2 and OsMADS4 may arise from protein divergence or from differences in their expression patterns within lodicule and stamen whorls. In this study, we have examined the dynamic expression pattern of both rice PI-like genes and have examined the likelihood of their functional redundancy for lodicule development. We show OsMADS2 transcripts occur at high levels in developing lodicules and transcripts are at reduced levels in stamens. In fully differentiated lodicules, OsMADS2 transcripts are more abundant in the distal and peripheral regions of lodicules, which are the tissues that are severely affected in OsMADS2 knock-down florets (Prasad and Vijayraghavan, 2003). The onset of OsMADS4 expression is in very young floret meristems before organ primordia emergence and this is expressed before OsMADS2. In florets undergoing organogenesis, high level OsMADS4 expression occurs in stamens and carpels and transcripts are at low level in lodicules (Yadav, Prasad and Vijayraghvan, 2007). Thus, we show that these paralogous genes differ in the onset of their activation and their stable transcript distribution within lodicules and stamens that are the conserved expression domains for PI-like genes. Since the expression of OsMADS4 in OsMADS2 knock-down florets is normal, our results show OsMADS2 has unique functions in lodicule development. Thus our data show subfunctionalization of these paralogous rice PI-like genes. To identify target genes regulated by OsMADS2 that could contribute to lodicule differentiation, we have adopted whole genome transcript analysis of wild-type and dsRNAiOsMADS2 panicles with developing florets. This analysis has identified potential down-stream targets of OsMADS2 many of which encode transcription factors, components of cell division cycle and signalling factors whose activities likely control lodicule differentiation. The expression levels of few candidate targets of OsMADS2 were examined in various floret organs. Further, the spatial expression pattern for four of these down-stream targets of OsMADS2 was analysed and we find overlap with OsMADS2 expression domains (Yadav, Prasad and Vijayraghvan, 2007). The predicted functions of these OsMADS2 target genes can explain the regulation of growth and unique vascular differentiation of this short fleshy modified petal analog. OsMADS1, a rice E-class gene, is a master regulator of other transcription factors and auxin and cytokinin signalling pathways In Arabidopsis four redundant SEPALLATA factors (E-class) are co-activators of other floral organ fate determining MADS-domain factors (classes ABCD) and thus contribute to floral meristem and floral organ development (Krizek and Fletcher, 2005). Among the grass-specific sub-clade of SEP-like genes, rice OsMADS1 is the best characterized. Prior studies in our lab showed that OsMADS1 is expressed early throughout the floret meristem before organ primordia emergence and later is restricted to the developing lemma and palea primordia with weak expression in carpel (Prasad et al, 2001). Stable expression continues in these floret organs. OsMADS1 plays critical non-redundant functions to specify a determinate floret meristem and also regulates floret organ identities (Jeon et al., 2000; Prasad et al, 2001; 2005; Agarwal et al., 2005; Chen et al., 2006). In the present study, we have adopted two different functional genomic approaches to identify genes down-stream of OsMADS1 in order to understand its mechanism of action during floret development. We have studied global transcript profiles in WT and dsRNAiOsMADS1 panicles and find OsMADS1 is a master regulator of a significant fraction of the genome’s transcription factors and also a number of genes involved in hormone-dependent cell signalling. We have validated few representative genes for transcription factors as targets regulated by OsMADS1. In a complementary approach, we have determined the consequences of induced-ectopic over-expression of a OsMADS1:ΔGR fusion protein in shoot apical meristems of transgenic plants. Transcript levels for candidate target genes were assessed in induced tissues and compared to mock-treated meristems and also with meristems induced for OsMADS1:ΔGR but blocked for new protein synthesis. These analyses show that OsMADS55 expression is directly regulated by OsMADS1. Importantly, OsMADS55 is related to SVP that plays an important role in floral transition and floral meristem identity in Arabidopsis. OsHB3 and OsHB4, homeodomain transcription factors, with a probable role in meristem function, are also directly regulated by OsMADS1. The regulation of such genes by OsMADS1 can explain its role in floret meristem specification. In addition to regulating other transcription factors, OsMADS1 knock-down affects expression of genes encoding proteins in various steps of auxin and cytokinin signalling pathways. Our differential expression profiling showed OsMADS1 positively regulates the auxin signalling pathway and negatively regulates cytokinin mediated signalling events. Through our induced ectopic expression studies of OsMADS1:ΔGR, we show OsMADS1 directly regulates the expression of OsETTIN2, an auxin response transcription factor, during floret development. Overall, we demonstrate that OsMADS1 modulates hormonal pathways to execute its functions during floret development on the spikelet meristems. Functional studies of OsMGH3; an auxin-responsive indirect target of OsMADS1 To better understand the contribution of auxin signalling during floret development, we have functionally characterized OsMGH3, a down-stream indirect target of OsMADS1, which is a member of the auxin-responsive GH3 family. The members of this family are direct targets of auxin response factors (ARF) class of transcription factors. GH3-proteins inactivate cellular auxin by conjugating them with amino acids and thus regulate auxin homeostasis in Arabidopsis (Staswick et al., 2005). OsMGH3 expression in rice florets overlaps with that of OsMADS1 (Prasad et al, 2005). In this study, we have demonstrated the consequences of OsMGH3 over-expression and knock-down. The over-expression of OsMGH3 during vegetative development causes auxin-deficient phenotypes such as dwarfism and loss of apical dominance. Its over-expression in developing panicles that was obtained by driving its expression from tissue-specific promoters created short panicles with reduced branching. The latter is a phenotype similar to that observed upon over-expression of OsMADS1. In contrast, the down-regulation of endogenous OsMGH3 through RNA-interference produced auxin over-production phenotypes such as ectopic rooting from aerial nodes. Knock-down of OsMGH3 expression in florets affected carpel development and pollen viability both of which affect floret fertility. Taken together, this study provides evidence for the importance of auxin homeostasis and its transcriptional regulation during rice panicle branching and floret organ development. Our analysis of various conserved transcription factors during rice floret development suggest that factors like OsMADS2, OsMADS4 and OsMADS1 are master regulators of gene expression during floret meristem specification and organ development. The target genes regulated by these factors contribute to development of morphologically distinct rice florets.