Gotowa bibliografia na temat „OsMADS4”

The MADS (MCM1-AGAMOUS-DEFFICIENS-SRF) gene family has a preserved domain called MADS-box that regulates downstream gene expression as a transcriptional factor. Reports have revealed three MADS genes in rice, OsMADS62, OsMADS63, and OsMADS68, which exhibits preferential expression in mature rice pollen grains. To better understand the transcriptional regulation of pollen germination and tube growth in rice, we generated the loss-of-function homozygous mutant of these three OsMADS genes using the CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9) system in wild-type backgrounds. Results showed that the triple knockout (KO) mutant showed a complete sterile phenotype without pollen germination. Next, to determine downstream candidate genes that are transcriptionally regulated by the three OsMADS genes during pollen development, we proceeded with RNA-seq analysis by sampling the mature anther of the mutant and wild-type. Two hundred and seventy-four upregulated and 658 downregulated genes with preferential expressions in the anthers were selected. Furthermore, downregulated genes possessed cell wall modification, clathrin coat assembly, and cellular cell wall organization features. We also selected downregulated genes predicted to be directly regulated by three OsMADS genes through the analyses for promoter sequences. Thus, this study provides a molecular background for understanding pollen germination and tube growth mediated by OsMADS62, OsMADS63, and OsMADS68 with mature pollen preferred expression.

Abstract Unlike many eudicot species, grasses have duplicated PI/GLO-like genes. Functional analysis of one of the rice PI/GLO paralogs, OsMADS2, is reported here. Our data demonstrate its essential role in lodicule development and implicate the second PI/GLO paralog, OsMADS4, to suffice for stamen specification. We provide the first evidence for differential contributions of grass PI/GLO paralogs in patterning second- and third-whorl floral organs.

Abstract The APETALA1 (AP1)/FRUITFULL (FUL)-like transcription factor OsMADS18 plays diverse functions in rice development, but the underlying molecular mechanisms are far from fully understood. Here, we report that down-regulation of OsMADS18 expression in RNAi lines caused a delay in seed germination and young seedling growth, whereas the overexpression of OsMADS18 produced plants with fewer tillers. In targeted OsMADS18 genome-edited mutants (osmads18-cas9), an increased number of tillers, altered panicle size, and reduced seed setting were observed. The EYFP-OsMADS18 (full-length) protein was localized to the nucleus and plasma membrane but the EYFP-OsMADS18-N (N-terminus) protein mainly localized to the nucleus. The expression of OsMADS18 could be stimulated by abscisic acid (ABA), and ABA stimulation triggered the cleavage of HA-OsMADS18 and the translocation of OsMADS18 from the plasma membrane to the nucleus. The inhibitory effect of ABA on seedling growth was less effective in the OsMADS18-overexpressing plants. The expression of a set of ABA-responsive genes was significantly reduced in the overexpressing plants. The phenotypes of transgenic plants expressing EYFP-OsMADS18-N resembled those observed in the osmads18-cas9 mutants. Analysis of the interaction of OsMADS18 with OsMADS14, OsMADS15, and OsMADS57 strongly suggests an essential role for OsMADS18 in rice development.

The ALOG domain genes, named after the Arabidopsis LSH1 and Oryza G1 (ALOG) proteins, have frequently been reported as key developmental regulators in rice and Arabidopsis. However, the investigation of the ALOG gene family is limited. Here, we conducted a genome-wide investigation of the ALOG gene family in rice and six other species. In total, eighty-four ALOG domain genes were identified from the seven species, of which fourteen ALOG domain genes (OsG1/G1Ls) were identified in the rice genome. The fourteen OsG1/G1Ls were unevenly distributed on eight chromosomes, and we found that eight segmental duplications contributed to the expansion of OsG1/G1Ls in the rice genome. The eighty-four ALOG family genes from seven species were classified into six clusters, and the ALOG domain-defined motifs 1, 2, and 3 were highly conserved across species according to the phylogenetic and structural analysis. However, the newly identified motifs 4 and 5 were only present in monocots, indicating a specified function in monocots. Moreover, OsG1/G1Ls exhibited tissue-specific expression patterns. Coexpression analysis suggested that OsG1 integrates OsMADS50 and the downstream MADS-box genes, such as OsMADS1, to regulate the development of rice inflorescence; OsG1L7 potentially associates with OsMADS22 and OsMADS55 to regulate stem elongation. In addition, comparative expression analysis revealed the conserved biological functions of ALOG family genes among rice, maize, and Arabidopsis. These results have shed light on the functional study of ALOG family genes in rice and other plants.

Flowering is a key agronomic trait that influences adaptation and productivity. Previous studies have indicated the genetic complexity associated with the flowering response in a photoinsensitive weedy rice accession PSRR-1 despite the presence of a photosensitive allele of a key flowering gene Hd1. In this study, we used whole-genome and RNA sequencing data from both cultivated and weedy rice to add further insights. The de novo assembly of unaligned sequences predicted 225 genes, in which 45 were specific to PSRR-1, including two genes associated with flowering. Comparison of the variants in PSRR-1 with the 3K rice genome (RG) dataset identified unique variants within the heading date QTLs. Analyses of the RNA-Seq result under both short-day (SD) and long-day (LD) conditions revealed that many differentially expressed genes (DEGs) colocalized with the flowering QTLs, and some DEGs such as Hd1, OsMADS56, Hd3a, and RFT1 had unique variants in PSRR-1. Ehd1, Hd1, OsMADS15, and OsMADS56 showed different alternate splicing (AS) events between genotypes and day length conditions. OsMADS56 was expressed in PSRR-1 but not in Cypress under both LD and SD conditions. Based on variations in both sequence and expression, the unique flowering response in PSRR-1 may be due to the high-impact variants of flowering genes, and OsMADS56 is proposed as a key regulator for its day-neutral flowering response.

Le riz (Oryza sativa L) est la source principale d'alimentation pour plus de la moitié de la population mondiale (Khush, 2005). La production de riz devrait augmenter de plus de 40% en 2030 pour satisfaire la demande de croissance de la population. Chaque année, environ 25% de la production est perdue à cause des insectes ravageurs, des maladies et des mauvaises herbes (Khus, 2005). Des pertes semblables sont dues aux stress abiotiques comme la sécheresse. L'objectif de mon travail de thèse a consisté à étudier la fonction de deux facteurs de transcription (FT) à boîte MADS OsMADS25 et OsMADS26 dans la réponse aux stress ou dans le développement. Pour cela, j'ai généré des lignées de riz surexprimant les ADNc codant ces FT et aussi des lignées interférées pour le gène OsMADS26 en utilisant deux GST différentes pour induire de l'ARN interférence destiné à détruire les ARNm OsMADS26. Dans le cas du gène OsMADS25 qui appartient à un groupe de cinq gènes phylogénétiquement proches, j'ai généré des plantes exprimant la protéine OsMADS25 fusionnée avec le motif répresseur dominant de la transcription EAR. Les lignées T2 exprimant le FT OsMADS25 fusionné au motif EAR présentent un phénotype semblable à celui d'une lignée d'insertion de TDNA dans ce gène. Ces plantes sont caractérisées par une forte réduction du nombre de leur talle et par une hauteur plus importante de la talle principale. Les plantes qui surexpriment OsMADS25 natif ne présentent pas de phénotype particulier. Ceci suggère que le gène OsMADS25 pourrait être impliqué dans la régulation du nombre de talles chez le riz bien qu'il soit exprimé au niveau de la racine. Le mode d'action du gène OsMADS25 sur le contrôle du développement des méristèmes axillaire du riz reste à préciser. Les lignées interférées OsMADS26 présentent une meilleure résistance à Magnaporthe oryzae (Mo) et à Xanthomonas oryzae pv. Oryzae (Xoo), deux principaux pathogènes du riz, et aussi une meilleure capacité de restauration après l'application d'un stress hydrique par rapport aux lignées témoin tandis que les lignées surexprimant OsMADS26 sont plus sensibles à ces stress. Les analyses de QPCR et du transcriptome que nous avons effectuées ont mis en évidence l'expression constitutive plus élevée dans les lignées interférées de plusieurs gènes de réponse aux stress biotique et abiotique. Ces résultats suggèrent que OsMADS26 pourrait être un inhibiteur général des mécanismes de défense de la plante et que les plantes interférée OsMADS26 sont dans un statut physiologique de type primed-like qui leur permettent d'être plus résistantes aux stress. Les lignées interférées pour OsMADS26 sont très peu affectées dans leur développement. Le gène OsMADS26 est donc un gène très intéressant pour les programmes d'amélioration du riz
MADS-box transcription factors (TF) have been mostly characterized for their involvement of plant development such as floral organogenesis and flowering time. Some of them are involved in stress related developmental processes such as abscission, fruit ripening and senescence. Overexpression of the rice OsMADS26 TF suggested a function in stress response. Here we report that OsMADS26 interfered lines presented a better resistance against two major pathogens of rice, Xanthomonas oryzae (Xoo) and Magnaportae oryzae (Mo) and a better recovery capacity after a water stress period. Transcriptome analysis revealed that several biotic and abiotic stresses related genes were up regulated in OsMADS26 interfered lines. In addition QPCR analysis showed that the expression of a set of biotic and abiotic genes was induced when OsMADS26 interfered lines were infected by Xoo or submitted to a water stress. This indicated that OsMADS26 is a negative regulator of biotic and abiotic stress response in rice. Taking in account the data previously published that showed that inducible overexpression of OsMADS26 resulted in the activation of expression of genes involved in jasmonic acid or reactive oxygen species biosynthesis, we postulate that OsMADS26 may be a hub regulator of stress response in rice and that it may be posttranscriptional regulated to modulate negatively or positively rice response to various stresses.In addition we have shown in this thesis that an insertion mutant line disrupting the OsMADS25 gene is characterized by a reduced number of tiller. This phenotype was also obtained in transgenic lines expressing the OsMADS25 transcription factor fused with a dominant motif inhibitor of transcription. Thissuggested that OsMADS25 is involved in the control of tiller development in rice.Key words: Rice, stress, blast, tillering, MADS-box, transcription factor, OsMADS26, OsMADS25, transcriptome

Organs in modern dicot flowers are positioned in concentric rings (whorls). The outermost whorl has green protective sepals, internal to which are showy petals, and the reproductive stamen and carpel whorls. Florets of rice, cereals and grasses evolved certain morphological and functionally distinct features in their non-reproductive organs. Striking among them are the highly modified petal analogs; called lodicules, and the large bract-like outermost organs named palea and lemma. The analogy of these modified rice floret organs to sepals and petals is debated. The two lodicules of rice florets are small (limited growth in Proximal-Distal axis), thick (extensive growth in Dorsal-Ventral axis) and are asymmetrically positioned to occupy only one half of the second whorl. They perform an important mechanical role in the partial opening of the flower for stamen emergence and subsequent closing. Their asymmetric position, small fleshy structure with many parenchymatous cell layers and their regulated physiology for swelling and collapse are critical for these functions. Understanding the developmental mechanisms of these organs that underlie their function is of direct interest to evo-devo plant biologists and breeding programs aimed at crop yield improvement. Given these implications, for a deeper understanding of plant development and potential future uses in crop breeding, we define the objectives of this study and report our key findings Objective 1: Uncovering the gene targets of rice class B PISTILLATA-like (PI-like) factors that regulate lodicule and stamen development Objective 2: Characterizing an OsMADS2 target gene; AP2/EREBP86 encoding AINTEGUMENTA-like/PLETHORA (AIL/PLT) family transcription factor. Overall, this study expands our knowledge on traits and molecular mechanisms controlled by rice class B PI-like factors: OsMADS2 and OsMADS4, and provides new insights on their functional divergence that greatly extend our understanding of lodicule and stamen development. This study sheds light on some molecular mechanisms triggered by AP2/EREBP86 that can initiate and maintain shoot meristem fate which have the potential to improve somatic embryogenesis.

碩士
國立中興大學
分子生物學研究所
105
M52048, a T-DNA insertion mutant, shows dwarf, early flowering, node bending and impaired in panicle exsertion and it has been demonstrated to have three flanking genes OsMADS14, OsMADS34 and OsCP7 activated. Transgenic plants, Ubi:OsMADS14, ectopically overexpressing OsMADS14 revealed dwarf and early flowering phenotype, and up-regulated expression of florigen genes Hd3a and RFT1 were observed in the previous study. The first part of this study is to investigate the possible mechanisms that how the expression of Hd3a and RFT1 can be up-regulated by OsMADS14. At first, the expression profile of rice florigen regulators such as OsPRR37, OsCO3, DTH2, OsDof12, PPS and RFL were analyzed. Among them, the expression of RFL was promoted and OsCO3 was repressed suggesting that the activation of Hd3a and RFT1 might result from the up-regulation of RFL and/or down-regulation of OsCO3. Secondly, the phase transition miRNAs, miR156 and miR172 were investigated and results showed the expression of miR156 was slightly activated while miR172 was repressed suggesting that the delayed phase transition might occurred in Ubi:OsMADS14. Thirdly, the expression of potential floral repressors such as putative OsTOE1 (TARGET OF EAT1), putative OsTEM1 (OsTEMPRANILLO1), putative OsTEM2 and RCN1 that may regulate by OsMADS14 were analyzed. Results showed that OsTEM1 and RCN1 were activated, suggesting the possible involvement of partial counteraction of the effect of early flowering by OsMADS14. Finally, physiological functions of these putative floral repressors were studied by investigating their correspondent T-DNA insertion activation mutants such as M59289 (for OsTOE1), M78020 (for RCN1) and M89461 (for OsTEM1). Among these mutants, only the putative OsTEM1 gene in mutant M89461 was activated and a slightly late flowering phenotype was observed in mutant M89461 as well suggesting that the putative OsTEM1 might be a floral repressor in rice. Transgenic plants, Ubi:OsMADS34, ectopically overexpressing OsMADS34 revealed impaired panicle exsertion phenotype and that resulted by the reduced length of the first internode was previously demonstrated. The second part of this study is to investigate the possible mechanisms that how the length of the first internode was significant reduced by overexpressing OsMADS34. Results of the microarray assays comparing Ubi:OsMADS34 to TNG67 revealed differential expressed of many stress-related genes. Among them, 31 genes designated as M1 to M31 were selected for further study and their expressions were to be confirmed by RT-PCR. Results showed that the expression profile of M1, M2, M4, M8, M9, M10, M16, M17, M18, M21, M22, M23, M24, M26, M27, M30 and M31 were in accordance with the results of microarray assays suggesting that the expression of these genes were regulated by overexpression of OsMADS34.

碩士
國立中興大學
分子生物學研究所
99
A rice T-DNA insertion mutant M0052048 showing extreme early flowering, bending tillers and impairment in panicle exertion was isolated from the Taiwan Rice Insertion Mutant (TRIM) library. This mutant contained a copy of the T-DNA tag inserted in the chromosome number 3 at the 64,867 bp position of OSJBa0032E21 BAC clone where the expression of 048-3 (OsMADS34), 048-4 (OsMADS14) and 048-7 (putative cysteine proteinase, OsCP7) genes were activated by the 35S enhancer located in the T-DNA. The OsMADS14 and OsMADS34 are MADS box-containing transcription factors that have important roles in plant growth and development and furthermore, OsCP7 is a putative cysteine proteinase that may be involved in the program cell death, pollen and xylem maturation, embryogenesis and flowering time in plants. In order to understand the functions of these activated genes and their contribution to the mutant phenotype, transgenic rice over-expressing each of these genes were created and expression profiles of these genes in various rice plant tissues were analyzed. RT-PCR analysis revealed that OsMADS14 and OsMADS34 expressed mainly in panicles but not necessarily in vegetative tissues, with the exception of a relatively high expression level of OsMADS14 in the 90-day-old leaf; For OsCP7, no clear expression signals were detected in all tested tissues. Transgenic rice Ubi:MADS14 showed constitutive expression of OsMADS14 in leaf tissue and revealed extreme early (63.6 days vs 121.3 days in TNG67) flowering and tiller bending phenotypes. The transgenic rice Ubi:MADS34 showed constitutive expression of OsMADS34 and revealed impairment in panicle exertion and slightly early (110 days vs 121.3 days) flowering compared to TNG67. For the overexpression study of OsCP7, no transgenic plant with ubiqutin promoter construct (Ubi:CP7) was obtained, thus a 1.6 kb promoter region from native OsCP7 gene was used to replace the ubiqutin promoter and several ectopically-expressed OsCP7 transgenic rice plants were obtained. These OsCP7 transgenic rice plants showed increased levels of OsCP7 mRNA and protein and higher cysteine protease activity compare to that of TNG67. In addition, these plants had reduced height, approximately 88% of TNG67, and revealed brown lesions on the surfaces of most spikelets that were neither observed in the TNG67 nor in M0052048. In summary, the present study suggests that the activation of OsMADS14 and OsMADS34 genes contributes to early flowering, bending tillers and impairment in panicle exertion phenotypes in the mutant M0052048. However, the effect of OsCP7 activation in mutant M0052048 and the function of OsCP7 in rice plants are still not clear and further investigations will be needed to answer these questions.

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

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

碩士
國立中興大學
分子生物學研究所
103
The T-DNA mutant M52048 identified from Taiwan Rice Insertional Mutant (TRIM) library showed dwarf, early flowering, node bending and impaired in panicle exertion. Three flanking genes, OsMADS34, OsMADS14 and OsCP7 (putative cysteine protease 7) were activated in this mutant. Both OsMADS34 and OsMADS14 belong to MADS-box gene family that may participate in regulation of flowering time and the identity of floral organ. OsCP7 is a member of C1A cysteine proteases. In this study, the function of OsMADS14 and OsCP7 were further investigated. Previous study has demonstrated that over-expression of OsMADS14 could cause early flowering. To understand whether or not any other flowering regulatory genes were affected by the expression of OsMADS14. The flowering regulatory genes including OsGI, OsMADS50, Ehd2, Hd1, Ehd1, Hd3a, RFT1, OsMADS14 and OsMADS18 were investigated in mutant M52048 and Ubi:OsMADS14, Ubi:OsMADS34 and OsCP7:OsCP7 transgenic rice respectively. Results showed two florigen genes, Hd3a and RFT1, expressed much earlier in M52048 and Ubi:OsMADS14 but not in Ubi:OsMADS34 and OsCP7:OsCP7, suggesting that Hd3a and RFT1 were regulated by the expression of OsMADS14. The mechanism how the expression of OsMADS14 could regulate florigen genes requires further investigation. Expression of OsCP7 driven by the maize ubiquitin promoter or the CaMV 35S promoter in transgenic rice cannot be obtained successfully. However transgenic rice, OsCP7:OsCP7 using 1.6 kb of OsCP7 promoter could be easily obtained and OsCP7:OsCP7 transgenic rice plants revealed slightly shorter in plant height, delayed flowering, lower fertility and lesion-like spots on spikelet. In contrast to the wild-type where no OsCP7 was detected in panicles, the RNA and protein expressions of OsCP7 in OsCP7:OsCP7 transgenic rice were detected in leaves at all development stages and panicles, and their expressions in transgenic rice correlated to the observed phenotypes. In addition, the phenotypes of segregated homozygous plants showed more significant than those of heterozygous plants within the same transgenic line, suggesting the dosage effect of transgene. However the expression levels of RNA and protein cannot be differentiated between homo- and hetero-zygous lines. To unravel the causes that lead to lower fertility of OsCP7:OsCP7, the floral organ and pollen viability were investigated. The floral organ showed no obvious differences between wild-type and OsCP7:OsCP7, but the pollen viability of OsCP7:OsCP7 was lower than that of wild-type, indicating that continuing expression of OsCP7 influence pollen development. Further investigation also indicated that the lesion-like spots on spikelet was correlated with the expression levels of OsCP7 and the lesions could possibly due to the programmed cell death caused by the activity of increased mature OsCP7 present in spikelet.

碩士
輔英科技大學
生物科技系碩士班
100
Oncidium is one of the most important orchids used for cut flowers and potted plants in Taiwan. The creation of new cultivars with novel traits is important for Oncidium to enhance the competitiveness of orchid industry. However, traditional breeding processes are limited by the long life cycle and self-incompatibility. The objectives of this study intend to analyze rice AP1-like gene OsMADS14 in Arabidopsis and Oncidium through genetic transformation. In this study, the rice gene OsMADS14 driven by maize ubiqutin promoter was introduced into Arabidopsis and Oncidium Gower Ramsey mediated by Agrobacterium tumefaciens. Twenty one independent ubiquitin::OsMADS14 transgenic Arabidopsis plants were produced, confirmed by PCR and RT-PCR analysis. The ectopic expression of OsMADS14 in transgenic Arabidopsis plants showed dwarf, early flowering and terminal flowers. In addition, the OsMADS14 gene were introduced into Oncidium Gower Ramsey using Agrobacterium tumefaciens-mediated transformation, protocorm-like bodies ( PLBs ) of Oncidium were used as explants materials for genetic transformation and selected on medium containing 5ppm hygromycin. The resistant transgenic Oncidium were analyzed by PCR, RT-PCR and histochemical GUS assay, indicating the transgene integrated into the genome. The resistant transgenic Oncidium showed leave bending phenotype. The results of this study suggest that the rice OsMADS14 gene could be applied early-flowering and dwarf traits to ornamental flowers.

碩士
國立中興大學
分子生物學研究所
103
The T-DNA mutant M52048 identified from Taiwan Rice Insertional Mutant (TRIM) library showed dwarf, early flowering, node bending and impaired in panicle exertion. Three flanking genes, OsMADS34, OsMADS14 and OsCP7 (putative cysteine protease 7) were activated in this mutant. Both OsMADS34 and OsMADS14 belong to MADS-box gene family that may participate in regulation of flowering time and the identity of floral organ. OsCP7 encode a putative cysteine protease, belongs to C1A cysteine protease family, its function remains unknown. In this study the function of OsMADS34 and OsCP7 were further investigated. Previous study showed that over-expression of OsMADS34, Ubi:OsMADS34 transgenic rice, could cause slightly early flowering and impaired in panicle exertion. Morphological dissection indicated that the impaired in panicle exertion was mainly caused by shortening the first internode (peduncle). Inhibition of the peduncle elongation caused by drought stress and ABA accumulation has been reported. In the present study, some drought-related genes, such as DREB1A, DREB1E and EATB, were regulated in Ubi:OsMADS34 transgenic rice and therefore hypothesized that the phenotype of transgenic rice may regulated by drought stress or plant hormones. However, treated transgenic rice with ABA inhibitor and/or GA could not improve the peduncle elongation and panicle exertion, suggested that the shortened peduncle and impaired in panicle exertion in Ubi:OsMADS34 transgenic rice might not cause by ABA accumulation. Analysis of GA biosynthesis related genes and the cell elongation promoting genes, at the internodes, revealed high expression levels of EATB, EUI1, GA13ox1, GA20ox2, OsPK1 and lower expression of XTH28, suggested that the shortened internode might due to the imbalance expression of these genes. However the mechanism how these genes involved in internode elongation remain to be elucidated. In addition to the shortened peduncle, the anther development was also affected in Ubi:OsMADS34, suggesting that OsMADS34 function as an E class MADS-box gene may interact with other MADS-box genes to regulate the floral organ development. Further study by searching OsMADS34 interaction proteins will help us to unravel the possible function of OsMADS34. For the study of OsCP7 gene, we were unable to obtained stable transgenic rice lines with constitutive promoter constructs, suggesting that constitutively ectopic expression of OsCP7 might cause lethal. Instead, transgenic lines with a 1.6 kb of OsCP7 promoter construct, OsCP7:OsCP7, were successfully obtained. OsCP7:OsCP7 revealed slightly dwarf, delayed flowering, lesion-like spots on panicles and lower fertility, and these phenotypes are correlated to the expression of OsCP7 gene. The possible mechanisms that cause these aberrant panicle developments were under investigated.

The Czech avant-garde artist Bohumil Kubišta came from a rural farming family. Educated in Hradec Králové, Kubišta moved to Prague in 1903 to attend art school, first at the School for Applied Art (1903–4) and then at the Academy of Fine Art (1904–5). After a year of service in 1905 with the Austrian Imperial Navy in Pula he studied at the Institute of Fine Art in Florence (1906–7). Upon his return to Prague he contributed to the first exhibition of Osma (The Eight, spring 1907) before returning to military duty in Pula. In summer 1908 he participated in Osma’s second and final exhibition.