Thèses sur le sujet « Inflorescences architecture »

Zea mays L. is one of the world’s most agronomically important crop. The understanding of the molecular basis of inflorescences architecture and seed development may be useful for agronomic purposes. The major goal of this research is to investigate different aspect of maize development to shed light on the genetic mechanisms involved in the formation of maize inflorescences as well as seed development. In the first part of my thesis, the mechanisms regulating inflorescences development have been investigated by studying a new barren mutant, barren inflorescence173 (bif173). The recessive mutant bif173 is affected in the formation of axillary meristems, showing defects in inflorescences development, such as a reduction in the number of spikelets and branches in the tassel and smaller and more disorganized ears. The phenotype of this mutant is not fully penetrant and its severity seems to be related to temperature or light changes. Also, we demonstrated that bif173, like other barren mutants, is involved in auxin biology and may play a role in auxin signaling. In order to identify the gene responsible of bif173 mutation, a RNA-seq analysis was carried out to closely examine a mapping region previously identified and one SNP present only in bif173 mutant transcripts was found. This SNP represents a non-synonymous mutation in the coding region of the gene GRMZM2G038401, causing a change of a very conserved amino acid in the encoded protein. This gene encodes a metalloprotease, homologous to the FtsH ATP- dependent metalloproteases, a conserved family of membrane- bound proteases. The ubiquitous localization of the GRMZM2G038401 transcripts seems to be consistent with the numerous functions of these proteases. As evidence that GRMZM2G038401 gene is a good candidate for bif173 mutation is the fact that the SNP found in the RNA-seq reads was not present in teosinte and other maize inbred lines, suggesting that it is not a polymorphism due to the genetic variability among maize background. In order to confirm that GRMZM2G038401 is the gene responsible for bif173 mutation, plants homozygous for a transposon insertion are currently growing and if the phenotype resembles the bif173 mutant phenotype, this gene will be confirmed as the causative gene. This finding will shed light on the molecular mechanisms regulating inflorescences development in maize and will increase our knowledge in auxin biology. In the second part of my thesis, genetic mechanisms acting in seed development have been investigated, particularly focusing on gametogenesis and embryogenesis. In A. thaliana, DME is a gene encoding a DNA glycosylase/lyase, active in the central cell of the female gametophyte before fertilization. The role of this enzyme is essential for the viability of the seed, in fact, acting as a demethylase, it activates the expression of maternal alleles, establishing imprinting in the endosperm. Here, two DME homologues in maize were identified: ZmDME1 and ZmDME2. The proteins encoded by these genes showed a high homology with A. thaliana DME and a conserved protein structure characteristic of the DME family. A phylogenetic analysis also suggested that these proteins have a common evolutionary origin. The expression of these genes was found in different stages of gametogenesis, previously identified through a morphological analysis. ZmDME1 and ZmDME2 showed a different expression pattern compared to A. thaliana DME, i.e. the expression was not only found in the mature gametophyte containing the central cell, but also in the embryo and endosperm and in all the vegetative tissues tested. Furthermore, the localization of the expression of ZmDME1 and ZmDME2 in the mature gametophyte was detected not only in the central cell but also in the other cells of the embryo sac and in the nucellus. In A. thaliana dme mutants produce non viable seeds, with enlarged endosperm and aborted embryos. A functional analysis using Zmdme1 mutant plants revealed no defects in vegetative and reproductive phases, producing all normal-shaped seeds. A morphological analysis of these mutants showed that gametogenesis and embryogenesis occur normally. Nevertheless, further analyses are needed to verify the function of these genes. Even if the lack of DME orthologues in monocots has been previously hypothesized, recent findings suggest that a similar mechanism of DNA demethylation may take place in monocot gametophyte. Thus, we discuss about the possibility that ZmDME1 and ZmDME2 may be responsible of active demethylation in maize gametophyte, allowing the proper development of embryo and endosperm.

[ES] Las leguminosas son un grupo de plantas consideradas de gran importancia por su valor nutricional para la alimentación humana y ganadera. Además, las familias de leguminosas se caracterizan por rasgos distintivos de desarrollo como su inflorescencia compuesta y su compleja ontogenia floral. Para comprender mejor estas características distintivas, es importante estudiar los genes reguladores clave involucrados en el desarrollo de la inflorescencia y la flor. El gen SUPERMAN (SUP) es un factor transcripcional de dedos de zinc (Cys2-Hys2) considerado como un represor activo que controla el número de estambres y carpelos en A. thaliana. Además, SUP está involucrado en la terminación del meristemo floral y el desarrollo de los tejidos derivados del carpelo. El objetivo principal de este trabajo fue la caracterización funcional del ortólogo de SUP en la leguminosa modelo Medicago truncatula (MtSUP). Logramos este objetivo en base a un enfoque de genética reversa, análisis de expresión génica y ensayos de complementación y sobreexpresión. Nuestros resultados muestran que MtSUP es el gen ortólogo de SUP en M. truncatula. MtSUP comparte algunos de los roles ya descritos para SUP con algunas variaciones. Curiosamente, MtSUP controla la determinación del meristemo inflorescente secundario (I2) y de los primordios comunes (CP) a pétalos y estambres. Por tanto, MtSUP controla el número de flores y de pétalos-estambres que producen el meristemo I2 y los primordios comunes, respectivamente. MtSUP muestra funciones novedosas para un gen de tipo SUP, desempeñando papeles clave en los meristemos que confieren complejidad de desarrollo a esta familia de angiospermas. Este trabajo permitió identificar a MtSUP, un gen clave que forma parte de la red reguladora genética que subyace al desarrollo de la inflorescencia compuesta y de las flores en la leguminosa modelo M. truncatula.
[CA] Les lleguminoses són un gran grup de plantes considerades de gran importància pel seu valor nutricional per a l'alimentació humana i ramadera. A més, les famílies de lleguminoses es caracteritzen per trets distintius de desenrotllament com la seua inflorescència composta i la seua complexa ontogènia floral. Per a comprendre millor estes característiques distintives, és important estudiar els gens reguladors clau involucrats en la inflorescència i el desenrotllament floral. El gen SUPERMAN (SUP) és un factor transcripcional de dits de zinc (Cys2-Hys2) considerat com un repressor actiu que controla el nombre d'estams i carpels en A. thaliana. A més, SUP està involucrat en la terminació del meristemo floral i el desenrotllament dels teixits derivats del carpel. "L'objectiu principal d'este treball va ser la caracterització funcional de l'ortòleg de SUP en la lleguminosa model Medicago truncatula (MtSUP) . Aconseguim l'objectiu amb base en un enfocament genètic invers, anàlisi d'expressió gènica i assajos de complementació i sobreexpressió. Els nostres resultats mostren que MtSUP és el gen ortòleg de SUP en M. truncatula. MtSUP compartix alguns dels rols ja descrits per a SUP amb variacions. Curiosament, MtSUP està involucrat en la determinació del meristemo de la inflorescència secundària (I2) i els primordios comuns (CP). Per tant, MtSUP controla el nombre de flors i pètals-estams que produïxen el meristemo I2 i els primordios comuns, respectivament. MtSUP mostra funcions noves per a un gen tipus SUP, exercint papers clau en els meristemos que conferixen complexitat de desenrotllament a esta família d'angiospermes. "Este treball va permetre identificar a MtSUP, un gen clau que forma part de la xarxa reguladora genètica darrere de la inflorescència composta i el desenrotllament de flors en la lleguminosa model M. truncatula.
[EN] Legumes are a large group of plants considered of great importance for their nutritional value in human and livestock nutrition. Besides, legume families are characterized by distinctive developmental traits as their compound inflorescence and complex floral ontogeny. For a better understanding of these distinctive features is important to study key regulatory genes involved in the inflorescence and floral development. The SUPERMAN (SUP) gene is a zinc-finger (Cys2-Hys2) transcriptional factor considered to be an active repressor that controls the number of stamens and carpels in A. thaliana. Moreover, SUP is involved in the floral meristem termination and the development of the carpel marginal derived tissues. The main objective of this work was the functional characterization of the SUP orthologue in the model legume Medicago truncatula (MtSUP). We achieved this objective based on a reverse genetic approach, gene expression analysis, and complementation and overexpression assays. Our results show that MtSUP is the orthologous gene of SUP in M. truncatula. MtSUP shares some of the roles already described for SUP with variations. Interestingly, MtSUP controls the determinacy of the secondary inflorescence (I2) meristem and the common primordia (CP). Thus, MtSUP controls the number of flowers and petal-stamens produced by the I2 meristem and the common primordia respectively. MtSUP displays novel functions for a SUP-like gene, playing key roles in the meristems that confer developmental complexity to this angiosperm family. This work allowed to identify MtSUP, a key gene that participates in the genetic regulatory network underlying compound inflorescence and flower development in the model legume M. truncatula.
I would like to thanks the Spanish Ministry of Economy and Competitiveness for the grant (MINECO; BIO2016-75485-R) that supported this work. Special thanks to the Generalitat Valenciana for funding my doctorate with the Santiago Grisolía predoctoral scholarships
Rodas Méndez, AL. (2021). MtSUPERMAN controls the number of flowers per inflorescence and floral organs in the inner three whorls of Medicago truncatula [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/171474
TESIS

During angiosperm evolution, innovations in vegetative and reproductive organs have resulted in tremendous morphological diversity, which has played a crucial role in the ecological success of flowering plants. Morindeae (Rubiaceae) display considerable diversity in growth form, inflorescence architecture, flower size, and fruit type. Lianescent habit, head inflorescence, small flower, and multiple fruit are the predominant states, but arborescent habit, non-headed inflorescence, large flower, and simple fruit states occur in various genera. This makes Morindeae an ideal model for exploring the evolutionary appearances and transitions between the states of these characters. We reconstructed ancestral states for these four traits using a Bayesian approach and combined nuclear/chloroplast data for 61 Morindeae species. The aim was to test three hypotheses: 1) self-supporting habit is generally ancestral in clades comprising both lianescent and arborescent species; 2) changes from lianescent to arborescent habit are uncommon due to "a high degree of specialization and developmental burden''; 3) head inflorescences and multiple fruits in Morindeae evolved from non-headed inflorescences and simple fruits, respectively. Lianescent habit, head inflorescence, large flower, and multiple fruit are inferred for Morindeae, making arborescent habit, non-headed inflorescence, small flower, and simple fruit derived within the tribe. The rate of change from lianescent to arborescent habit is much higher than the reverse change. Therefore, evolutionary changes between lianescent and arborescent forms can be reversible, and their frequency and trends vary between groups. Moreover, these changes are partly attributed to a scarcity of host trees for climbing plants in more open habitats. Changes from large to small flowers might have been driven by shifts to pollinators with progressively shorter proboscis, which are associated with shifts in breeding systems towards dioecy. A single origin of dioecy from hermaphroditism is supported.

Inflorescence architecture is a key agronomical trait that determines fruit and seed yield. Understanding the genetic basis of inflorescence architecture will not only contribute to elucidate crop evolution/domestication mechanisms but also improve crop grain yield. Flowering plants develop different types of inflorescences, such as racemes in Arabidopsis and panicles in rice. The architecture is established during the early stages of reproductive development and it is determined by the activity of different meristem types and by the timing of the transition between indeterminate meristems to determinate ones. Inflorescence development is finely regulated by a genetic network that includes meristem identity genes and genes that regulate their expression; many genes are already known but others have still to be characterized to provide insight into how this complex process is controlled. Transcriptomic analysis performed in rice and in Arabidopsis through laser microdissection of different meristematic tissues highlighted differentially expressed genes belonging to the ALOG family suggesting their role in inflorescence patterning. We focus on G1L1, G1L2, and G1L5 of rice and on LSH1, LSH3, and LSH4 of Arabidopsis. G1L5 is already known to be a major regulator of inflorescence architecture, whereas LSH3 and LSH4 seem to have a role in meristem maintenance and organogenesis. Their expression profiles were analyzed by qRT-PCR and RNA in situ hybridization experiments using meristematic tissues from both species. We are also generating single and double/triple K.O mutants in different combinations by CRISPR-Cas9 genome editing technology to have a better understanding of their role in inflorescence patterning. The data so far obtained demonstrate the role of G1L1 and G1L2 in inflorescence branching and spikelet number determination and we also propose a role for G1L2 in root development. Furthermore, LSH1 seems to be involved in meristem maintenance and organ differentiation, and LSH3 in stem elongation. We propose the hypothesis that LSH1, LSH3, and LSH4 play a redundant function in inflorescence development.

The development of the wheat inflorescence, or spike, determines the number, size and shape of grain produced. Altering spike development has the potential to increase grain yield to support increasing global demands. Despite the importance of this specialized structure, little is known about the genes which underlie its development and thus contribute to grain output. Yield potential in wheat can be modified by numerous developmental outcomes. The number of vegetative branches, known as tillers determines the number of spikes per plant. Similarly, the number of nodes within the spike determines the number of grain bearing structures called spikelets. Increasing the number of spikelets per node in the form of branching further increases yield potential. Finally, the fertility of florets, which are the grain bearing units found within spikelets determine the number of grain set. Optimising these inflorescence structures is key for maximising yield potential in wheat. The important floral regulators, PPD-1 and FT positively regulate floral transition and influence the rate of floral development. Increased expression of PPD-1 leads to increases in FT expression and promotion of floral development. We have shown that loss-of-function mutations in both PPD-1 and FT contribute to a reduction in expression of floral meristem identity genes, a delay in flowering, an increase in nodes within the inflorescence and the production of a unique form of inflorescence branch called a paired spikelet. The important maize domestication gene TB1 is responsible for a reduction in vegetative branching and the single stem structure of modern maize. We show that increased copies of wheat TB1 reduce vegetative branches known as tillers as well as increase paired spikelet formation in wheat. We propose that TB1 interacts with FT in the inflorescence causing a similar reduction in meristem identity genes as we observed in FT and Ppd-1 mutants. The wheat domestication gene Q, an AP2 transcription factor, is one of only a handful of genes which is known to directly influence spike development in wheat. We have identified a novel gain-of-function mutation of Q which confers resistance to miR172, a microRNA that typically targets AP2 transcripts for degradation. We show that increased Q levels are associated with several phenotypes including, delayed flowering, the formation of ectopic florets in place of glumes, the formation of paired spikelets and a reduction in internode elongation throughout the plant. AP2 transcription factors are reported to delay flowering in other plant species. Increased expression of Q is therefore the likely cause of a delay in flowering time and the formation of paired spikelets not dissimilar to what we observed in loss-of-function mutations of FT and PPD-1. Taken together our results highlight a complex network of genes which regulate the number of grain producing units contributing to wheat yield. Many more genes regulating inflorescence development in wheat remain to be elucidated. With the recent release of the first wheat reference genome sequence, increased availability of reverse genetic resources and access to genome editing tools, it will be possible to identify novel gene function in wheat.

Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2008.
Source: Dissertation Abstracts International, Volume: 69-05, Section: B, page: 2757. Adviser: Torbert R. Rocheford. Includes bibliographical references. Available on microfilm from Pro Quest Information and Learning.

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.

The olive (Olea europaea L.) is one of the most ancient fruit trees and has been cultivated for its oil in the Mediterranean area for thousands of years. Today, the consumption of olive oil and table olives is increasing both in traditional producing countries and the entire world. Most olive cultivars are self-incompatible and do not produce a commercial yield after self pollination. In this thesis, inflorescence architecture and sexual compatibility relationships of some olive cultivars, and gene expression in olive pistils during flowering were studied. To study the inflorescence architecture of olive, 45 inflorescences in each of the cultivars Manzanillo, Mission, and Frantoio were checked every morning from flower opening to petal fall. The flower position on the inflorescence had a highly significant effect on the opening day in all cultivars. Terminal flowers and the flowers located on the primary branches opened earlier than flowers located on the secondary branches. Flower position also had a highly significant effect on gender in Manzanillo and Mission. In Manzanillo, the secondary branches had fewer perfect flowers than the primary branches. In Mission, the secondary branches had no perfect flowers at all. In Manzanillo, perfect flowers had significantly longer petal persistence than staminate flowers. To study flower competition within the inflorescence, the distal halves, on which the flowers tend to be perfect, of 120 inflorescences in three trees of Manzanillo were removed about one month before full bloom. This resulted in a highly significant increase in the percentage of perfect flowers on the proximal halves. The effects of shoot orientation and inflorescence location on inflorescence characteristics in the cultivars Frantoio, Kalamata, and Koroneiki were also studied. For each cultivar, inflorescence characteristics in three sections of shoots (top, middle, and base) and four sides of the three selected trees (north, south, east, and west) were recorded. The statistical analysis showed that basal inflorescences were shorter and with fewer flowers but with the same percentage of perfect flowers. Shoot orientation did not have any influence on these characteristics in any of the cultivars. Sexual compatibility was assessed using two methods. In the first method, controlled crossings were performed in the cultivars Frantoio, Koroneiki, and Kalamata. The pistils were harvested one week after hand pollination and stained with 0.1% aniline blue. The styles and ovules were separated, mounted in 80% glycerol, and observed under a fluorescence microscope. In Frantoio and Koroneiki, the number of ovules penetrated by a pollen tube was used to estimate the level of sexual compatibility. In Kalamata, the numbers of ovules penetrated by pollen tubes were not significantly different between treatments; therefore, the number of pollen tubes in the lower style was used. All the cultivars studied were self- incompatible. Frantoio (as a host) was incompatible with Koroneiki and Barnea but partially compatible with Mission. Koroneiki (as a host) was incompatible with Barnea but partially compatible with Frantoio and Mission. Kalamata (as a host) was compatible with Barnea, incompatible with Mission and Koroneiki in 2004, but partially compatible with them in 2005. In the second method, eight microsatellite markers were used for genotyping three Kalamata mother trees, 40 embryos per mother tree, and all the potential pollen donors. Genotyping data were analysed using FaMoz software, and the number of embryos assigned to each putative pollen donor was determined. Paternity analysis showed that Kalamata (as a host) was self-incompatible, compatible with Barnea, Benito, and Katsourela, but incompatible with Arbequina, Azapa, and Picual. To study the gene expression in olive pistils during flowering, a genomic approach was initiated using cDNA subtractive array analysis. Total RNA was isolated from olive pistils at two developmental stages, where self-incompatibility (SI) genes are expected to be differentially expressed: 1) small green flower buds (expression of SI genes not expected) and 2) large white flower buds containing receptive pistils just prior to opening (expression of SI genes expected). From each stage, cDNA libraries were prepared and put through forward and reverse subtractive hybridisations to enrich for differentially expressed cDNAs in stage 2. Macroarrays were prepared by printing 2304 differentially expressed cDNAs onto nylon membranes and hybridised with forwardand reverse-subtracted probes. The analysis identified 90 up-regulated cDNA clones highly expressed in receptive pistils. Further subtracted and unsubtracted hybridisations confirmed up-regulation of the majority of these cDNAs. Gene expression profiles across different tissues showed that most of the genes were pistil-specific. The expression pattern of the genes showed high similarity in Kalamata, Frantoio, Barnea, and Pendolino. All the screened genes were sequenced and their similarities were searched in the NCBI database. The most redundant and interesting up-regulated clones were those similar to a receptor protein kinase-like protein. Some versions of this protein play a role in the sporophytic SI system of Brassica and the gametophytic SI system of Papaver and rye.
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Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008

The olive (Olea europaea L.) is one of the most ancient fruit trees and has been cultivated for its oil in the Mediterranean area for thousands of years. Today, the consumption of olive oil and table olives is increasing both in traditional producing countries and the entire world. Most olive cultivars are self-incompatible and do not produce a commercial yield after self pollination. In this thesis, inflorescence architecture and sexual compatibility relationships of some olive cultivars, and gene expression in olive pistils during flowering were studied. To study the inflorescence architecture of olive, 45 inflorescences in each of the cultivars Manzanillo, Mission, and Frantoio were checked every morning from flower opening to petal fall. The flower position on the inflorescence had a highly significant effect on the opening day in all cultivars. Terminal flowers and the flowers located on the primary branches opened earlier than flowers located on the secondary branches. Flower position also had a highly significant effect on gender in Manzanillo and Mission. In Manzanillo, the secondary branches had fewer perfect flowers than the primary branches. In Mission, the secondary branches had no perfect flowers at all. In Manzanillo, perfect flowers had significantly longer petal persistence than staminate flowers. To study flower competition within the inflorescence, the distal halves, on which the flowers tend to be perfect, of 120 inflorescences in three trees of Manzanillo were removed about one month before full bloom. This resulted in a highly significant increase in the percentage of perfect flowers on the proximal halves. The effects of shoot orientation and inflorescence location on inflorescence characteristics in the cultivars Frantoio, Kalamata, and Koroneiki were also studied. For each cultivar, inflorescence characteristics in three sections of shoots (top, middle, and base) and four sides of the three selected trees (north, south, east, and west) were recorded. The statistical analysis showed that basal inflorescences were shorter and with fewer flowers but with the same percentage of perfect flowers. Shoot orientation did not have any influence on these characteristics in any of the cultivars. Sexual compatibility was assessed using two methods. In the first method, controlled crossings were performed in the cultivars Frantoio, Koroneiki, and Kalamata. The pistils were harvested one week after hand pollination and stained with 0.1% aniline blue. The styles and ovules were separated, mounted in 80% glycerol, and observed under a fluorescence microscope. In Frantoio and Koroneiki, the number of ovules penetrated by a pollen tube was used to estimate the level of sexual compatibility. In Kalamata, the numbers of ovules penetrated by pollen tubes were not significantly different between treatments; therefore, the number of pollen tubes in the lower style was used. All the cultivars studied were self- incompatible. Frantoio (as a host) was incompatible with Koroneiki and Barnea but partially compatible with Mission. Koroneiki (as a host) was incompatible with Barnea but partially compatible with Frantoio and Mission. Kalamata (as a host) was compatible with Barnea, incompatible with Mission and Koroneiki in 2004, but partially compatible with them in 2005. In the second method, eight microsatellite markers were used for genotyping three Kalamata mother trees, 40 embryos per mother tree, and all the potential pollen donors. Genotyping data were analysed using FaMoz software, and the number of embryos assigned to each putative pollen donor was determined. Paternity analysis showed that Kalamata (as a host) was self-incompatible, compatible with Barnea, Benito, and Katsourela, but incompatible with Arbequina, Azapa, and Picual. To study the gene expression in olive pistils during flowering, a genomic approach was initiated using cDNA subtractive array analysis. Total RNA was isolated from olive pistils at two developmental stages, where self-incompatibility (SI) genes are expected to be differentially expressed: 1) small green flower buds (expression of SI genes not expected) and 2) large white flower buds containing receptive pistils just prior to opening (expression of SI genes expected). From each stage, cDNA libraries were prepared and put through forward and reverse subtractive hybridisations to enrich for differentially expressed cDNAs in stage 2. Macroarrays were prepared by printing 2304 differentially expressed cDNAs onto nylon membranes and hybridised with forwardand reverse-subtracted probes. The analysis identified 90 up-regulated cDNA clones highly expressed in receptive pistils. Further subtracted and unsubtracted hybridisations confirmed up-regulation of the majority of these cDNAs. Gene expression profiles across different tissues showed that most of the genes were pistil-specific. The expression pattern of the genes showed high similarity in Kalamata, Frantoio, Barnea, and Pendolino. All the screened genes were sequenced and their similarities were searched in the NCBI database. The most redundant and interesting up-regulated clones were those similar to a receptor protein kinase-like protein. Some versions of this protein play a role in the sporophytic SI system of Brassica and the gametophytic SI system of Papaver and rye.
Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2008