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

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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.

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.