The objectives of this research were to examine factors influencing de novo flowering of Nicotiana on 2-3 x 10mm explants consisting of epidermal and 3-6 layers of subjacent cells (thin cell layers, TCLs) and to compare de novo to in vivo flowers. TCLs from short-day and long-day tobacco plants were compared with TCLs from day-neutral species to examine in vitro floral photoinduction and graft transmissibility of floral promoters and inhibitors. TCLs from photoperiodic species of tobacco did not form flowers de novo , whereas TCLs from day-neutral plants did flower. When TCLs were removed from photoperiodic plants and grafted in vitro to TCLs from day-neutral plants, there was no indication that a floral-promoter or inhibitor was transported through the non-vascular graft union. In vitro photoinduction of TCLs removed from photoperiodic plants was not possible under conditions conducive to in vitro flowering of TCLs from day-neutral species. TCLs taken from intraspecific F₁ and F₂ hybrids between short-day and day-neutral cultivars of N. tabacum were examined to assess the importance of genotype and photoperiod to de novo flowering. Flowering of the F₂ population occurred over a 9 week period under naturally decreasing photoperiod. Photoperiodic response and in vitro flowering were correlated in the F₂ population with fewer flowers produced per TCL with increasing short-day reaction. F₂ segregates whose TCLs did not yield de novo flowers were found among both day-neutral and short-day phenotypes. When de nova flowers were compared to in vivo flowers of diploid (2n=4x=48) N. tabacum 'Samsun' and haploid (2n=2x=24) plants derived from 'Samsun' anther culture, major morphological differences were found. Flower and anther sizes were reduced in de novo flowers and the numbers of anthers and pistils produced per flower were variable. TCLs from haploid plants produced more flowers in a shorter period of time than TCLs from diploid plants. Anthers cultured from de novo haploid plants were embryogenetic resulting in mixoploid plants; anthers from in vivo haploid flowers were not embryogenetic. Anthers from in vivo diploid plants were five times more embryogenetic than anthers from either de novo haploid or diploid flowers. Meiotic analysis revealed similar abnormalities from both in vivo and de novo microsporogenesis of haploids. / Ph. D.

Thesis (Ph. D.)--Ohio State University, 2007.

Orientadores: Marcos Antonio Machado, Marcio Alves Ferreira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-21T12:49:19Z (GMT). No. of bitstreams: 1 Mafra_ValeriaSiqueira_D.pdf: 24593212 bytes, checksum: 99883266609f22224577609d713195c0 (MD5) Previous issue date: 2012 / Resumo: O desenvolvimento floral envolve uma rede complexa de genes que agem no meristema apical para especificar a identidade do meristema floral e, mais adiante, a identidade de órgãos florais. Outros genes controlando a simetria e polaridade dos órgãos, assim como a influência de giberelinas e auxinas, desempenham um papel importante no estabelecimento do tamanho final e arquitetura da flor. Este trabalho propôs avaliar as alterações no transcriptoma do meristema durante a indução e diferenciação floral e em diferentes fases do desenvolvimento do botão floral em laranja doce. Para isto, foi construído um chip de microarranjo de citros contendo cerca de 32 mil unigenes de laranja doce, presentes no banco de dados CitEST. Para se avaliar o perfil global de expresssão, foram coletadas gemas em dois estágios de desenvolvimento, quatro estágios de desenvolvimento do botão floral e flores abertas. As análises estatísticas foram feitas por meio da comparação 2x2, considerando um dado estágio em relação ao estágio anterior. Foram encontrados 3.590 unigenes nãoredundantes com diferença de expressão em pelo menos uma das comparações e a análise de enriquecimento de ontologias (GSEA) definiu os processos biológicos mais representativos. Foram identificados vários unigenes de citros envolvidos com a sinalização do florescimento, regulação e morfogênese de órgãos florais. A validação por PCR em tempo real (RT-qPCR) foi feita com 29 genes candidatos e o perfil de expressão mostrou a mesma tendência do microarranjo. Para normalizar o nível de expressão dos genes alvo no RT-qPCR, foram usados os normalizadores GAPC2 e SAND. Esses genes foram selecionados como bons genes de referência em uma avaliação sistemática comparando a estabilidade de expressão de 15 genes em diferentes condições experimentais . As discussões sobre o possível papel dos genes e vias na regulação do desenvolvimento floral em citros são apresentadas. Uma vez que vários reguladores chave do florescimento são proteínas da família MADS-box, foi proposto identificar os genes MADS-box nos genomas de laranja doce e Clementina e avaliar o relacionamento filogenético entre o MADS-box de citros e Arabidopsis. As análises dos genomas de laranja doce e Clementina revelaram 83 e 92 genes MADS-box, respectivamente. Os MADS-box tipo II de citros foram distribuídos dentre as sub-famílias de MIKCc e MIKC*, enquanto que os genes do tipo I em geral não agruparam com os MADS-box de Arabidopsis. Este estudo espera obter uma maior compreensão sobre os eventos moleculares relacionados com o florescimento de citros e fornecer as bases para análises funcionais para descobrir o papel desses genes / Abstract: Flower development involves a complex gene network that acts in the shoot apical meristem to specify the floral meristem identity and, later, to determine the floral organ identity. Other genes controlling symmetry and polarity of floral organs, as well as the influence of gibberellins and auxins, play important roles in establishing the final size and architecture of the flower. This work aimed to evaluate changes in the meristem's transcriptome during floral induction and differentiation, and in different stages of flower bud development in sweet orange. For this purpose, we built a citrus microarray chip containing about 32,000 unique assemblies (unigenes) of sweet orange, present in the CitEST database. To assess the global expression profile, we collected buds at two developmental stages, bud flowers at four stages and open flowers. Statistical analyses were performed in a 2x2 comparison, taking into account a given developmental stage compared to the stage before. It was found 3,590 nonredundant unigenes that were differentially expressed at least one comparison and a Gene Set Enrichment Analysis defined the most representative biological processess. Several citrus unigenes differentially modulated involved with flowering signaling, regulation and floral organs morphogenesis were identified. Validation by real time PCR (RT-qPCR) was performed to 29 candidate genes and the expression profile showed the same tendency of microarray. To normalize the expression level of the target genes, we used GAPC2 and SAND reference genes. These genes were selected as good reference genes in a systematic evaluation comparing the expression stability of 15 genes in different experimental conditions. Discussions about the possible role of the genes and pathways in the regulation of citrus flower development are presented. Since several key regulators of flowering are proteins belonging the MADS-box family, we proposed to identify MADS-box genes in the sweet orange and Clementine genomes and to evaluate the phylogenetic relationship between citrus and Arabidopsis MADS-box genes. Analysis of the sweet orange and mandarin genomes revealed 76 and 91 MADS-box genes, respectively. Type II MADS-box genes of citrus were grouped into MIKCc and MIKC* sub-families whereas tipo I MADS-box genes of citrus and Arabidopsis were grouped in separated clades. This work will help in the understanding of the genetic events related to citrus flowering and provide the basis for functional analyses to uncover the role of these genes / Doutorado / Genetica Vegetal e Melhoramento / Doutor em Genetica e Biologia Molecular

This study examined floral initiation in Rudbeckia hirta at the biochemical, cellular, and whole plant levels. Histological and histochemical examination of floral initiation revealed that the pattern of initiation followed closely that described in other species. The primary difference was in the length of time over which initiation and differentiation occurred. When subjected to limited inductive photoperiods, R. hirta responded with a delay in flowering if the plants were returned to short days (SD) before bract initiation. Increased exposure to long days (LD) increased stem height and enhanced floral development. A limited induction period of at least 8 LD allowed enough of the floral stimulus to be translocated to the meristem to cause no interruption in development even upon return to non-inductive conditions. An inhibition of development occurred only when plants were returned to SD before periclinal divisions in the pith rib meristem commenced after approximately 8 LD. Axillary bud development and final plant height were dependent on the number of inductive LD received. Polyamines have been linked to floral initiation and, in this study, were strongly correlated to the stage of floral initiation. As initiation progressed, the observed increases in putrescine and spermidine were followed by a decrease after 16 LD, the observed onset of floral development. This was contrary to that previously observed in SD plants but followed a pattern similar to that reported for cytokinin behavior. Exogenous cytokinins have been used to stimulate floral initiation in several species but Rudbeckia hirta did not respond to benzyladenine (BA) applied at the onset of LD. Floral initiation has been found to begin after six to eight LD and, in most species, BA was most effective when applied during initiation. In an attempt to increase uptake, BA was dissolved in dimethyl sulfoxide (DMSO). This did not enhance the effects of BA and, in fact, DMSO was found to be toxic at concentrations of 25% or more. / Ph. D.

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.