Gotowa bibliografia na temat „Floral developmental genetics”

The concepts of phylogeny and floral genetics play a crucial role in understanding the origin and diversification of flowers in angiosperms. Angiosperms evolved a great diversity of ways to display their flowers for reproductive success with variations in floral color, size, shape, scent, arrangements, and flowering time. The various innovations in floral forms and the aggregation of flowers into different kinds of inflorescences have driven new ecological adaptations, speciation, and angiosperm diversification. Evolutionary developmental biology seeks to uncover the developmental and genetic basis underlying morphological diversification. Advances in the developmental genetics of floral display have provided a foundation for insights into the genetic basis of floral and inflorescence evolution. A number of regulatory genes controlling floral and inflorescence development have been identified in model plants such as Arabidopsis thaliana and Antirrhinum majus using forward genetics, and conserved functions of many of these genes across diverse non-model species have been revealed by reverse genetics. Transcription factors are vital elements in systems that play crucial roles in linked gene expression in the evolution and development of flowers. Therefore, we review the sex-linked genes, mostly transcription factors, associated with the complex and dynamic event of floral development and briefly discuss the sex-linked genes that have been characterized through next-generation sequencing.

AbstractThe floral developmental pathway in Arabidopsis thaliana is composed of several interacting regulatory genes, including the inflorescence architecture gene TERMINAL FLOWER1 (TFL1), the floral meristem identity genes LEAFY (LFY), APETALA1 (AP1), and CAULIFLOWER (CAL), and the floral organ identity genes APETALA3 (AP3) and PISTILLATA (PI). Molecular population genetic analyses of these different genes indicate that the coding regions of AP3 and PI, as well as AP1 and CAL, share similar levels and patterns of nucleotide diversity. In contrast, the coding regions of TFL1 and LFY display a significant reduction in nucleotide variation, suggesting that these sequences have been subjected to a recent adaptive sweep. Moreover, the promoter of TFL1, unlike its coding region, displays high levels of diversity organized into two distinct haplogroups that appear to be maintained by selection. These results suggest that patterns of molecular evoution differ among regulatory genes in this developmental pathway, with the earlier acting genes exhibiting evidence of adaptive evolution.

Timing of flowering is a fundamental developmental transition that has great ecological and agricultural importance. For perennial plants, seasonal timing of bloom and anthesis, which is the ultimate stage of flowering, can be determined by the net effect of several preceding developmental steps: seasonal timing of floral initiation, rate and extent of floral development before winter dormancy, duration of dormancy, and rate of floral development after release from dormancy. In the domestic apple (Malus ×domestica), fruit production has generally favored cultivars that bloom relatively early in the season. However, floral tissues are easily damaged by freezing temperatures, and freeze injury is especially problematic in years when abnormally warm temperatures in early spring lead to rapid floral development. To facilitate identification of genes/alleles that govern bloom time, and that could add versatility to production systems for apple, we evaluated seasonal bloom time for accessions of M. ×domestica, wild apple species (Malus sp.), and Malus hybrids maintained in a large germplasm diversity collection.

Abstract The Arabidopsis flowering-repressor gene FLOWERING LOCUS C (FLC) is a developmental switch used to trigger floral induction after extended growth in the cold, a process termed vernalization. In vernalized plants, FLC becomes transcriptionally silenced through a process that involves an epigenetic mechanism. We identified recessive mutations designated vernalization independence (vip) that confer cold-independent flowering and suppression of FLC. These mutations also lead to developmental pleiotropy, including specific defects in floral morphology, indicating that the associated genes also have functions unrelated to flowering time. We identified the VIP3 gene by positional cloning and found that it encodes a protein consisting almost exclusively of repeated Trp-Asp (WD) motifs, suggesting that VIP3 could act as a platform to assemble a protein complex. Constitutive transgenic expression of VIP3 in vernalized plants is insufficient to activate FLC, and thus VIP3 probably participates in the regulation of FLC as one component of a more extensive mechanism. Consistent with this, genetic analyses revealed that the VIP loci define a functional gene class including at least six additional members. We suggest that VIP3 and other members of this gene class could represent a previously unrecognized flowering mechanism.

Erythronium japonicum Decne (Liliaceae) flowers in early spring after overwintering. Its sexual reproduction process includes an underground development process of floral organs, but the underlying molecular mechanisms are obscure. The present study is aimed at exploring the transcriptional changes and key genes involved at underground floral developmental stages, including flower primordium differentiation, perianth differentiation, stamen differentiation, and pistil differentiation in E. japonicum. Multistage high-quality transcriptomic data resulted in identifying putative candidate genes for underground floral differentiation in E. japonicum. A total of 174,408 unigenes were identified, 28,508 of which were differentially expressed genes (DEGs) at different floral developmental stages, while only 44 genes were identified with conserved regulation between different stages. Further annotation of DEGs resulted in the identification of 270 DEGs specific to floral differentiation. In addition, ELF3, PHD, cullin 1, SE14, ZSWIM3, GIGNATEA, and SERPIN B were identified as potential candidate genes involved in the regulation of floral differentiation. Besides, we explored transcription factors with differential regulation at different developmental stages and identified bHLH, FAR1, mTERF, MYB-related, NAC, Tify, and WRKY TFs for their potential involvement in the underground floral differentiation process. Together, these results laid the foundation for future molecular works to improve our understanding of the underground floral differentiation process and its genetic regulation in E. japonicum.

Abstract A new mutant of Arabidopsis thaliana that initiates flowering early and terminates the inflorescence with floral structures has been identified and named terminal flower2 (tfl2). While these phenotypes are similar to that of the terminal flower1 (tfl1) mutant, tfl2 mutant plants are also dwarfed in appearance, have reduced photoperiod sensitivity and have a more variable terminal flower structure. Under long-day and short-day growth conditions tfl1 tfl2 double mutants terminate the inflorescence without development of lateral flowers; thus, unlike tfl1 single mutants the double mutant inflorescence morphology is not affected by day length. The enhanced phenotype of the double mutant suggests that TFL2 acts in a developmental pathway distinct from TFL1. The complex nature of the tfl2 single mutant phenotype suggests that TFL2 has a regulatory role more global than that of TFL1. Double mutant analysis of tfl2 in combination with mutant alleles of the floral meristem identity genes LEAFY and APETALA1 demonstrates that TFL2 function influences developmental processes controlled by APETALA1, but not those regulated by LEAFY. Thus, the TFL2 gene product appears to have a dual role in regulating meristem activity, one being to regulate the meristem response to light signals affecting the development of the plant and the other being the maintenance of inflorescence meristem identity.

Observations of floral development indicate that floral organ initiation in pentapetalous flowers more commonly results in a medially positioned abaxial petal (MAB) than in a medially positioned adaxial petal (MAD), where the medial plane is defined by the stem and the bract during early floral development. It was proposed that the dominant MAB petal initiation might impose a developmental constraint that leads to the evolution of limited patterns of floral zygomorphy in Asteridae, a family in which the floral zygomorphy develops along the medial plane and results in a central ventral (CV) petal in mature flowers. Here, I investigate whether the pattern of floral organ initiation may limit patterns of floral zygomorphy to evolve in pentapetalous angiosperms. I analyzed floral diagrams representing 405 species in 330 genera of pentapetalous angiosperms to reconstruct the evolution of floral organ initiation and the evolution of developmental processes that give rise to floral zygomorphy on a phylogenetic framework. Results indicate that MAB petal initiation is the most common; it occupies 86.2% of diversity and represents the ancestral state of floral organ initiation in pentapetalous angiosperms. The MAD petal initiation evolved 28 times independently from the ancestral MAB petal initiation. Among the 34 independent originations of floral zygomorphy, 76.5% of these clades represent MAB petal initiation, among which only 47% of the clades result a CV petal in mature flowers. The discrepancy is explained by the existence of developmental processes that result in floral zygomorphy along oblique planes of floral symmetry in addition to along the medial plane. Findings suggest that although the early floral organ initiation plays a constraining role to the evolution of patterns of floral zygomorphy, the constraint diverges along phylogenetically distantly related groups that allow the independent originations of floral zygomorphy through distinct development processes in pentapetalous angiosperms. In additional study, the butterfly-like flowers of Schizanthus are adapted to pollination by bees, hummingbirds, and moths. I investigated the genetic basis of the zygomorphic corolla, for which development is key to the explosive pollen release mechanism found in the species of Schizanthus adapted to bee pollinators. I examined differential gene expression profiles across the zygomorphic corolla of Schizanthus pinnatus, a bee-pollinated species, by analyzing RNA transcriptome sequencing (RNA- seq). Data indicated that CYC2 is not expressed in the zygomorphic corolla of Sc. pinnatus, suggesting CYC2 is not involved in the development of floral zygomorphy in Schizanthus (Solanaceae). The data also indicated that a number of genes are differentially expressed across the corolla.

Il a déjà été montré chez plusieurs espèces que quelques gènes architectes (les gènes A, B et C) étaient responsables de l’identité des organes floraux : sépales, pétales, étamines et pistils. Cependant, les réseaux de gènes régulés en aval permettant le développement des organes floraux, et pouvant expliquer une partie de la diversité des plantes à fleurs demeurent peu connus. L’objectif de mon projet de thèse vise à étudier le développement de la fleur de façon plus détaillée avec comme modèle d’étude les fleurs de Petunia x hybrida. Mes travaux de thèse se sont organisés autour de plusieurs axes de recherche, en commençant par l’obtention du transcriptome spécifique aux pétales chez le Pétunia. La stratégie imaginée a été de réaliser un RNA-Seq sur de jeunes fleurs sauvages et sur une collection unique de mutants homéotiques. Les données obtenues nous ont permis d’identifier une liste de 452 gènes présentant un profil d’expression pétale spécifique. Afin d’identifier l’implication de ces gènes dans le développement des pétales, un crible de génétique inverse a été réalisé sur 98 gènes de la liste (95 mutants générés avec le système transposon et 3gènes avec le système CRISPR-Cas9). De façon étonnante, aucun phénotype pétale n’a pu être associé à la mutation des gènes candidats. Cependant dans la population générée pour le crible pétale, nous avons pu observer une population de plantes ségrégeant pour un phénotype avec des défauts dans le développement floral. Nous avons confirmé que ce phénotype n’était pas associé à un des gènes candidats initialement ciblé et par une méthode de génétique directe, nous avons identifié que ce phénotype était causé par une insertion de transposon dans un facteur de transcription R2R3-MYB. Dans un autre chapitre, j’ai ciblé plusieurs gènes avec de potentiels rôles dans le développement de la fleur avec la technique CRISPR-Cas9. Les mutations obtenues dans le gène classe-C PMADS3 ont contribué à une publication (Morel et al., 2018) ainsi qu’à la description de façon plus approfondie des fonctions des gènes de la classe-C chez le Pétunia. Dans la dernière partie, nous avons montré que selon la couche cellulaire où le gène DEF (responsable de l’identité des pétales) est exprimé, le pétale ne présente pas un phénotype sauvage. La croissance et la forme des pétales (tube versus limbe) nécessitent l’intervention et la coordination de plusieurs couches cellulaires (L1, L2 et L3)
While the master regulators of floral organ identity have been identified in multiple plantspecies, it remains poorly understood how the downstream transcriptional programs finally lead to the development of the different floral organs, and how evolutionary variations in these programs have yielded the astonishing floral architectural diversity existing in nature. The main objective of my PhD work was to start to address these fundamental questions by analysing floral development in Petunia x hybrida, chosen as model for its elaborate petal architecture combined with the availability of a powerful genetics toolkit. My research started with the identification of the petal transcriptome composition acting downstream of the homeotic genefunctions (Chapter 1). To achieve this, we used an RNAseq strategy on young flowers from a unique collection of floral homeotic mutants, complemented with wild-type samples. We finallyobtained a list of more than 400 potentially interesting genes involved in petal development. Toprovide a detailed analysis for Petunia petal development we used a reverse genetics approachand selected 95 genes expressed during petal development for functional analysis by transposonmutagenesis. I also introduced the CRISPR-Cas9 technology in the team (Chapter 3), targeting3 petal candidate genes for which no transposon insertions in their coding sequence were found. Unfortunately, we did not manage to find eye-catching defects in petal development linked tothe selected mutations. However, in the population generated for the petal reverse genetics screen we encountered a small family in which a mutation segregated causing a novel floral developmental defect strongly affecting petal and stamen development. We confirmed that this mutation was unrelated to the petal candidate gene initially targeted, and by a forward genetic approach we demonstrated that it was instead caused by a different transposon insertion in anR2R3-MYB transcription factor (Chapter 2). With the CRISPR-Cas9 technology I also targetedsome interesting genes involved in flower development like the C-class gene PMADS3. Iobtained KO mutants, and this result was part of a paper (Morel et al., 2018) and allowed a detailed description of the C-class genes function in petunia (Chapter 3). In the last part, we investigated how tube and limb development of Petunia petals depend on the cell-layer specificaction of a MADS-box transcription factor. This allowed to define the contribution of the differentcell-layers in petal development (Chapter 4). Put together, my PhD work should provide a better understanding of floral organ development and architectural diversity

Specialization on insect and animal pollinators is thought to be the driving force for the evolution of floral traits. Specifically in the New World (NW), the oil-bee pollination syndrome has led to the convergence of floral characters in two distantly related families of core eudicots, Malpighiaceae and Krameriaceae. Both families display a flag-like structure that establishes a zygomorphic flower and floral oil rewards in epithelial elaiophores. These traits work concomitantly to attract and reward female oil-bees that help fertilize these flowers and in return receive oils. The underlying genetics of floral zygomorphy were studied in several clades of core eudicots, which implicated CYCLOIDEA2-(CYC2-)like genes of the TCP gene family to play a role in the establishment and maintenance of this trait. In Malpighiaceae, previous work demonstrated that two CYC2-like genes, CYC2A and CYC2B, are expressed during development correlated with establishing zygomorphy in flowers of NW Malpighiaceae. In this thesis work, I investigated the underlying developmental and genetic basis for the establishment of non-homologous and yet functionally similar traits in the oil-bee pollinated flowers of Malpighiaceae and Krameriaceae. In Chapter 1, I investigated the modification of a conserved CYC2 genetic program in the Old World (OW) acridocarpoids of Malpighiaceae following a major biogeographic disjunction. And in Chapter 2, I studied the floral ontogeny and genetic basis of floral zygomorphy development in Krameria lanceolata Torrey of Krameriaceae. This thesis work sheds light on the significance of the interdisciplinary study of floral symmetry evolution through comparative pollination ecology, development, and genetics.

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.

abstract: The Cape Floral Region (CFR) in southwestern South Africa is one of the most diverse in the world, with >9,000 plant species, 70% of which are endemic, in an area of only ~90,000 km2. Many have suggested that the CFR's heterogeneous environment, with respect to landscape gradients, vegetation, rainfall, elevation, and soil fertility, is responsible for the origin and maintenance of this biodiversity. While studies have struggled to link species diversity with these features, no study has attempted to associate patterns of gene flow with environmental data to determine how CFR biodiversity evolves on different scales. Here, a molecular population genetic data is presented for a widespread CFR plant, Leucadendron salignum, across 51 locations with 5-kb of chloroplast (cpDNA) and 6-kb of unlinked nuclear (nuDNA) DNA sequences in a dataset of 305 individuals. In the cpDNA dataset, significant genetic structure was found to vary on temporal and spatial scales, separating Western and Eastern Capes - the latter of which appears to be recently derived from the former - with the highest diversity in the heart of the CFR in a central region. A second study applied a statistical model using vegetation and soil composition and found fine-scale genetic divergence is better explained by this landscape resistance model than a geographic distance model. Finally, a third analysis contrasted cpDNA and nuDNA datasets, and revealed very little geographic structure in the latter, suggesting that seed and pollen dispersal can have different evolutionary genetic histories of gene flow on even small CFR scales. These three studies together caution that different genomic markers need to be considered when modeling the geographic and temporal origin of CFR groups. From a greater perspective, the results here are consistent with the hypothesis that landscape heterogeneity is one driving influence in limiting gene flow across the CFR that can lead to species diversity on fine-scales. Nonetheless, while this pattern may be true of the widespread L. salignum, the extension of this approach is now warranted for other CFR species with varying ranges and dispersal mechanisms to determine how universal these patterns of landscape genetic diversity are.
Dissertation/Thesis
Ph.D. Biology 2013

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.

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.

According to the theory of Hipocrates (3rd century BC) "all diseases begin in the intestines". It is now known that intestinal microorganisms participate in physiological processes such as: immune system functioning, detoxification, inflammation, neurotransmitter and vitamin production, nutrient absorption, hunger, and satiety signaling, carbohydrate and fat burning. Thus, a beneficial microbial flora is maintained by proper nutrition. Also, in the literature, there are microbiome-specific associations with different pathologies: attention deficit hyperactivity disorder (ADHD), asthma, autism, allergies, chronic fatigue, depression, anxiety, and diabetes. To prevent these pathologies, in the children's growth and development it must be considered multiple factors: the type of birth (natural or caesarean), genetics, general health, physical activity, sedentarism, sleep quality, and appropriate nutrition.

The primary objectives for the US lab included: the characterization of ELF3 transcription and translation; the creation and characterization of various transgenic lines that misexpress ELF3; defining genetic pathways related to ELF3 function regulating floral initiation in Arabidopsis; and the identification of genes that either interact with or are regulated by ELF3. Light quality, photoperiod, and temperature often act as important and, for some species, essential environmental cues for the initiation of flowering. However, there is relatively little information on the molecular mechanisms that directly regulate the developmental pathway from the reception of the inductive light signals to the onset of flowering and the initiation of floral meristems. The ELF3 gene was identified as possibly having a role in light-mediated floral regulation since elj3 mutants not only flower early, but exhibit light-dependent circadian defects. We began investigating ELF3's role in light signalling and flowering by cloning the ELF3 gene. ELF3 is a novel gene only present in plant species; however, there is an ELF3 homolog within Arabidopsis. The Arabidopsis elj3 mutation causes arrhythmic circadian output in continuous light; however, we show conclusively normal circadian function with no alteration of period length in elj3 mutants in dark conditions and that the light-dependent arrhythmia observed in elj3 mutants is pleiotropic on multiple outputs regardless of phase. Plants overexpressing ELF3 have an increased period length in constant light and flower late in long-days; furthermore, etiolated ELF3-overexpressing seedlings exhibit a decreased acute CAB2 response after a red light pulse, whereas the null mutant is hypersensitive to acute induction. This finding suggests that ELF3 negatively regulates light input to both the clock and its outputs. To determine whether ELF3's action is phase dependent, we examined clock resetting by light pulses and constructed phase response curves. Absence of ELF3 activity causes a significant alteration of the phase response curve during the subjective night, and overexpression of ELF3 results in decreased sensitivity to the resetting stimulus, suggesting that ELF3 antagonizes light input to the clock during the night. Indeed, the ELF3 protein interacts with the photoreceptor PHYB in the yeast two-hybrid assay and in vitro. The phase ofELF3 function correlates with its peak expression levels of transcript and protein in the subjective night. ELF3 action, therefore, represents a mechanism by which the oscillator modulates light resetting. Furthermore, flowering time is dependent upon proper expression ofELF3. Scientifically, we've made a big leap in the understanding of the circadian system and how it is coupled so tightly with light reception in terms of period length and clock resetting. Agriculturally, understanding more about the way in which the clock perceives and relays temporal information to pathways such as those involved in the floral transition can lead to increased crop yields by enabling plants to be grown in suboptimal conditions.

Meristems were the central issue of our project. Genes that are required for cell division, cell elongation, cell proliferation and cell fate were studied in the tomato system. The analysis of the dUTPase and threonine deaminase genes, along with the dissection of their regulatory regions is completed, while that of the RNR2 and PPO genes is at an advanced stage. All these genes were isolated in our laboratory. In addition, 8 different MADS box genes were studied in transgenic plants and their genetic relevances discovered. We have also shown that a given MADS box gene can modify the polarity of cell division without affecting the fate of the organ. In vivo interaction between two MADS box genes was demonstrated and the functional dependency of the tomato agamous gene on the TM5 gene product established. We have exploited the Knotted1 meristematic gene in conjunction with tomato leaf meristematic genes to show that simple and compound leaves and, for that matter, sepals and compound leaves, are formed by two different developmental programs. In this context we have also isolated and characterized the tomato Knotted1 gene (TKnl) and studied its expression pattern. A new program in which eight different meristematic genes in tomato will be studied emerged as a result of these studies. In essence, we have shown that it is possible to study and manipulate plant developmental systems using reverse genetic techniques and have provided a wealth of new molecular tools to interested colleagues working with tomato. Similarly, genes responsible for cell division, cell proliferation and cell fate were studied in Arabidopsis floral meristems. Among these genes are the TSO1, TSO2, HANABA TARANU and UNUSUAL FLORAL ORGANS genes, each affecting in its own way the number of pattern of cell divisions, and cell fate, in developing Arabodopsis flowers. In addition, new methods have been established for the assessment of the function of regulatory gene action in the different clonal layers of developing floral meristems.

Objectives. The major aim of the proposed research was to establish an efficient and reproducible genetic transformation system for Easter lily and gladiolus using either biolistics or Agrobacterium. Transgenic plants containing pathogen-derived genes for virus resistance were to be developed and then tested for virus resistance. The proposal was originally aimed at studying cucumber mosaic virus (CMV) resistance in plants, but studies later included bean yellow mosaic virus (BYMV). Monoclonal antibodies were to be tested to determine their effectiveness in interning with virus infection and vector (aphid) transmission. Those antibodies that effectively interfered with virus infection and transmission were to be cloned as single chain fragments and used for developing transgenic plants with the potential to resist virus infection. Background to the topic. Many flower crops, as lily and gladiolus are propagated vegetatively through bulbs and corms, resulting in virus transmission to the next planting generation. Molecular genetics offers the opportunity of conferring transgene-mediated disease resistance to flower crops that cannot be achieved through classical breeding. CMV infects numerous plant species worldwide including both lilies and gladioli. Major conclusions, solutions and achievements. Results from these for future development of collaborative studies have demonstrated the potential transgenic floral bulb crops for virus resistance. In Israel, an efficient and reproducible genetic transformation system for Easter lily using biolistics was developed. Transient as well as solid expression of GUS reporter gene was demonstrated. Putative transgenic lily plantlets containing the disabled CMV replicase transgene have been developed. The in vitro ability of monoclonal antibodies (mAbs) against CMV to neutralize virus infectivity and block virus transmission by M. persicae were demonstrated. In the US, transgenic Gladiolus plants containing either the BYMV coat protein or antisense coat protein genes have been developed and some lines were found to be virus resistant. Long-term expression of the GUS reporter gene demonstrated that transgene silencing did not occur after three seasons of dormancy in the 28 transgenic Gladiolus plants tested. Selected monoclonal antibody lines have been isolated, cloned as single chain fragments and are being used in developing transgenic plants with CMV resistance. Ornamental crops are multi-million dollar industries in both Israel and the US. The increasing economic value of these floral crops and the increasing ban numerous pesticides makes it more important than ever that alternatives to chemical control of pathogens be studied to determine their possible role in the future. The cooperation resulted in the objectives being promoted at national and international meetings. The cooperation also enabled the technology transfer between the two labs, as well as access to instrumentation and specialization particular to the two labs.

The shoot apical meristem establishes plant architecture by continuously producing new lateral organs such as leaves, axillary meristems and flowers throughout the plant life cycle. This unique capacity is achieved by a group of self-renewing pluripotent stem cells that give rise to founder cells, which can differentiate into multiple cell and tissue types in response to environmental and developmental cues. Cell fate specification at the shoot apical meristem is programmed primarily by transcription factors acting in a complex gene regulatory network. In this project we proposed to provide significant understanding of meristem maintenance and cell fate specification by studying four transcription factors acting at the meristem. Our original aim was to identify the direct target genes of WUS, STM, KNAT6 and CNA transcription factor in a genome wide scale and the manner by which they regulate their targets. Our goal was to integrate this data into a regulatory model of cell fate specification in the SAM and to identify key genes within the model for further study. We have generated transgenic plants carrying the four TF with two different tags and preformed chromatin Immunoprecipitation (ChIP) assay to identify the TF direct target genes. Due to unforeseen obstacles we have been delayed in achieving this aim but hope to accomplish it soon. Using the GR inducible system, genetic approach and transcriptome analysis [mRNA-seq] we provided a new look at meristem activity and its regulation of morphogenesis and phyllotaxy and propose a coherent framework for the role of many factors acting in meristem development and maintenance. We provided evidence for 3 different mechanisms for the regulation of WUS expression, DNA methylation, a second receptor pathway - the ERECTA receptor and the CNA TF that negatively regulates WUS expression in its own domain, the Organizing Center. We found that once the WUS expression level surpasses a certain threshold it alters cell identity at the periphery of the inflorescence meristem from floral meristem to carpel fate [FM]. When WUS expression highly elevated in the FM, the meristem turn into indeterminate. We showed that WUS activate cytokinine, inhibit auxin response and represses the genes required for root identity fate and that gradual increase in WUCHEL activity leads to gradual meristem enlargement that affect phyllotaxis. We also propose a model in which the direction of WUS domain expansion laterally or upward affects meristem structure differently. We preformed mRNA-seq on meristems with different size and structure followed by k-means clustering and identified groups of genes that are expressed in specific domains at the meristem. We will integrate this data with the ChIP-seq of the 4 TF to add another layer to the genetic network regulating meristem activity.

Monoecious species such as melon and cucumber develop separate male and female (or bisexual) flowers on the same plant individual. They display complex genetic and hormonal regulation of sex patterns along the plant. Ethylene is known to play an important role in promoting femaleness and inhibiting male development, but many questions regarding critical sites of ethylene production versus perception, the relationship between ethylene and the sex determining loci, and the possible differences between melon and cucumber in this respect are still open. The general goal of the project was to elucidate the role of ethylene in determining flower sex in Cucumis species, melon and cucumber. The specific Objectives were: 1. Clone and characterize expression patterns of cucumber genes involved in ethylene biosynthesis and perception. 2. Genetic mapping of cloned genes and markers with respect to sex loci in melon and cucumber. 3. Produce and analyze transgenic melons altered in ethylene production or perception. In the course of the project, some modifications/adjustments were made: under Objective 2 (genetic mapping) a set of new mapping populations had to be developed, to allow better detection of polymorphism. Under Objective 3, cucumber transformation systems became available to us and we included this second model species in our plan. The main findings of our study support the pivotal role of ethylene in cucumber and melon sex determination and later stages of reproductive development. Modifying ethylene production resulted in profound alteration of sex patterns in melon: femaleness increased, and also flower maturation and fruit set were enhanced, resulting in earlier, more concentrated fruit yield in the field. Such effect was previously unknown and could have agronomic value. Our results also demonstrate the great importance of ethylene sensitivity in sex expression. Ethylene perception genes are expressed in sex-related patterns, e.g., gynoecious lines express higher levels of receptor-transcripts, and copper treatments that activate the receptor can increase femaleness. Transgenic cucumbers with increased expression of an ethylene receptor showed enhanced femaleness. Melons that expressed a defective receptor produced fewer hermaphrodite flowers and were insensitive to exogenous ethylene. When the expression of defective receptor was restricted to specific floral whorls, we saw that pistils were not inhibited by the blocked perception at the fourth whorl. Such unexpected findings suggest an indirect effect of ethylene on the affected whorl; it also points at interesting differences between melon and cucumber regarding the mode of action of ethylene. Such effects will require further study. Finally, our project also generated and tested a set of novel genetic tools for finer identification of sex determining genes in the two species and for efficient breeding for these characters. Populations that will allow easier linkage analysis of candidate genes with each sex locus were developed. Moreover, effects of modifier genes on the major femaleness trait were resolved. QTL analysis of femaleness and related developmental traits was conducted, and a comprehensive set of Near Isogenic Lines that differ in specific QTLs were prepared and made available for the private and public research. Marker assisted selection (MAS) of femaleness and fruit yield components was directly compared with phenotypic selection in field trials, and the relative efficiency of MAS was demonstrated. Such level of genetic resolution and such advanced tools were not used before to study these traits, that act as primary yield components to determine economic yields of cucurbits. In addition, this project resulted in the establishment of workable transformation procedures in our laboratories and these can be further utilized to study the function of sex-related genes in detail.

Structural and functional genomic approaches for marking and identifying genes that control chilling requirement in apricot and peach trees. Specific aims: 1) Identify and characterize the genetic nature of chilling requirement for flowering and dormancy break of vegetative shoots in Prunusgermplasm through the utilization of existing apricot (NeweYa'ar Research Center, ARO) and peach (Clemson University) genetic mapping populations; 2) Use molecular genetic mapping techniques to identify markers flanking genomic regions controlling chilling; 3) Comparatively map the regions controlling chilling requirement in apricot and peach and locate important genomic regions influencing chilling requirement on the Prunus functional genomic database as an initial step for identification of candidate genes; 4) Develop from the functional genomics database a set of markers facilitating the development of cultivars with optimized chilling requirements for improved and sustained fruit production in warm-winter environments. Dormant apricot (prunus armeniaca L.) and peach [Prunus persica (L.) Batsch] trees require sustained exposure to low, near freezing, temperatures before vigorous floral and vegetative bud break is possible after the resumption of warm temperatures in the spring. The duration of chilling required (the chilling requirement, CR) is determined by the climatic adaptation of the particular cultivar, thus limiting its geographic distribution. This limitation is particularly evident when attempting to introduce superior cultivars to regions with very warm winter temperatures, such as Israel and the coastal southern United States. The physiological mechanism of CR is not understood and although breeding programs deliberately manipulate CR in apricot and peach crosses, robust closely associated markers to the trait are currently not available. We used segregating populations of apricot (100 Fl individuals, NeweYa'ar Research Center, ARO) and peach (378 F2 individuals, Clemson University) to discover several discreet genomic loci that regulate CR and blooming date. We used the extensive genomic/genetic resources available for Prunus to successfully combine our apricot and peach genetic data and identify five QTL with strong effects that are conserved between species as well as several QTL that are unique to each species. We have identified markers in the key major QTL regions for testing in breeding programs which we are carrying out currently; we have identified an initial set of candidate genes using the peach physical/transcriptome map and whole peach genome sequences and we are testing these currently to identify key target genes for manipulation in breeding programs. Our collaborative work to date has demonstrated the following: 1) CR in peach and apricot is predominantly controlled by a limited number ofQTL loci, seven detected in a peach F2 derived map comprising 65% of the character and 12 in an apricot Fl map comprising 71.6% and 55.6% of the trait in the Perfection and A. 1740 parental maps, respectively and that peach and apricot appear in our initial maps to share five genomic intervals containing potentially common QTL. 2) Application of common anchor markers of the Prunus/peach, physical/genetic map resources has allowed us not only to identify the shared intervals but also to have immediately available some putative candidate gene information from these intervals, the EVG region on LG1 in peach the TALY 1 region in apricot on LG2 in peach; and several others involved in vernalization pathways (LGI and LG7). 3) Mapped BACcontigs are easily defined from the complete physical map resources in peach through the common SSR markers that anchor our CR maps in the two species, 4) Sequences of BACs in these regions can be easily mined for additional polymorphic markers to use in MAS applications.