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Ectopic expression of the floral homeotic gene AGAMOUS in transgenic Arabidopsis

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Abstract

The Arabidopsis floral homeotic gene AGAMOUS (AG) is required for development of the reproductive organs (stamens and carpels). In ag mutants, the loss of AG function leads to the conversion of these organs to the perianth organs (petals and sepals). In contrast, mutations in another floral homeotic gene, APETALA2 (AP2), result in the replacement of the perianth organs by the reproductive organs. On the basis of these observations, it has been proposed that AG and AP2 act in an antagonistic fashion. To test this hypothesis, we have studied the effects of ectopically expressed AG in transgenic Arabidopsis plants. The flowers of the transgenic plants exhibit a range of phenotypes mirroring those of ap2 mutants. These experiments provide direct evidence of the proposed antagonism between AG and AP2 functions, and the results strongly suggest that AG does indeed inhibit AP2 function.

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... The functions of these genes may be influenced by their expression levels. In Arabidopsis, weaker ectopic expression of C-class AGAMOUS (AG) in whorl 2 led to mild phenotypes and the petal identity was not changed (Mizukami & Ma 1992). In species other than Arabidopsis, B-class AP3/DEFICIENS and PISTILLATA/GLO-BOSA (PI/GLO) and C-class AG genes are functionally conserved, while the functions of AP1 and AP2 appear to be less conserved (Causier et al. 2010;Broholm et al. 2014). ...
... In Arabidopsis, the low level of ectopic expression of AG in whorl 2 is not able to change the identity of the petal to a stamen, and whether the stamen or petal develops is based on the relative expression level of AG (Mizukami & Ma 1992;Wollmann et al. 2010). Accordingly, we consider that there is a similar dose-dependency in C. indica, i.e. the C-class gene is expressed in the whole androecium, while fertile stamen identity is specified by relatively higher-level expression. ...
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• Canna indica is a common ornamental plant with asymmetric flowers decorated with colorful petaloid staminodes. The only fertile stamen comprises a one‐theca anther and a petaloid appendage, and represents the fewest stamen number in the order Zingiberales. The molecular mechanism for the asymmetric androecial petaloidy remains poorly understood. Here, we studied the identity specification in Canna stamen. • We observed four types of abnormal flowers concerning androecium identity transformations, and analyzed the corresponding floral symmetry changes. We further tested the expression patterns of B‐ and C‐class MADS‐box genes with in situ hybridization in normal Canna stamen. • Homeotic conversions in the androecium were accompanied by floral symmetry changes, and the asymmetric stamen is a key factor contributing to the floral asymmetry. Both B‐ and C‐class genes exhibited higher expression levels in anther primordium than other androecial parts. This kind of asymmetric expression pattern precisely corresponded to the asymmetric identities of Canna androecium. • We identified C. indica as a model species for studying the androecial organ identity and floral symmetry synthetically in Zingiberales, and hypothesized that homeotic genes specify floral organ identity in a putative dose‐dependent manner. These results add to the current understanding of organ identity‐related floral symmetry.
... M genes jointly regulate and form a flower development network. The AG of class plays an important role in the development of petals and stamens [47]. In A. tha mutants, almost no stamens exist because they transform into petals. ...
... Multiple genes jointly regulate and form a flower development network. The AG of class C gene plays an important role in the development of petals and stamens [47]. In A. thaliana ag mutants, almost no stamens exist because they transform into petals. ...
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AGAMOUS-LIKE 24 (AGL24) is a key gene regulating floral transition, but its involvement in flower organ identity remains largely unknown. In this study, we found that RhAGL24 is strongly related to petal and stamen development in rose. Its expression increases rapidly at the petal primordium development stage and maintains a high level until the complete differentiation stage. RhAGL24 silencing increases the number of malformed petals and decreases the number of stamens, indicating that this gene affects stamen petaloidy. RhAG (AGAMOUS), a class C gene associated with petal and stamen development, is downregulated in RhAGL24-silenced plants. Moreover, we found that RhAGL24 could directly bind to the promoter region of RhARF18 (AUXIN RESPONSE FACTORS 18), a regulator of RhAG. Our results suggested that RhAGL24-RhARF18 module regulates stamen petaloidy in rose and provide new insights into the function of AGL24 for plants.
... Based on their evolution of origin, plant MADS-box genes are classified into MIKC-type and M-type. Among the MIKC-type genes, AGAMOUS (AG) and SEPALLATA are the key regulators of reproductive developmental processes (i.e., flowering time, fruit and seed development) in model and non-model plants (Mizukami and Ma 1992;Pelaz et al. 2001;de Moura et al. 2017;Zhou et al. 2020). In Arabidopsis, members of AG subfamily are involved in the specification of floral reproductive organs and are required for the normal development of carpels, stamens and fruits Ma 1992, 1997;Dreni and Kater 2014). ...
... In Arabidopsis, members of AG subfamily are involved in the specification of floral reproductive organs and are required for the normal development of carpels, stamens and fruits Ma 1992, 1997;Dreni and Kater 2014). The loss of AG function in ag mutants of Arabidopsis results in the conversion of reproductive organs (stamens and carpels) to the perianth organs (petals and sepals) (Mizukami and Ma 1992). Overexpression of AG also induces early flowering and the curling of rosette leaves in Arabidopsis (Mizukami and Ma 1997). ...
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Main conclusion The expression of full-length cDNAs encoding lavender AGAMOUS-like (LaAG-like) and SEPALLATA3-like (LaSEP3-like) transcription factors induces early flowering and impacts the leaf morphology at a strong expression level in Arabidopsis. Abstract Lavandula angustifolia is widely cultivated as an ornamental plant due to its attractive flower structure, and as a source of valuable essential oils for use in cosmetics, alternative medicines, and culinary products. We recently employed RNA-Seq and transcript profiling to describe a number of transcription factors (TFs) that potentially control flower development in this plant. In this study, we investigated the roles of two TFs, LaAGAMOUS-like (LaAG-like) and LaSEPALLATA3-like (LaSEP3-like), that exhibited substantial homology to Arabidopsis thaliana floral development genes, AGAMOUS and SEPALLATA3, respectively, in flowering initiation in Arabidopsis. We stably and constitutively expressed LaAG-like and LaSEP3-like cDNAs in separate Arabidopsis plants. All transgenic plants flowered earlier than the wild-type controls. However, plants that modestly overexpressed the gene were phenotypically normal, while those that strongly expressed the transgene developed curly leaves. We also assessed the expression of five endogenous flowering time regulating genes, from which high expression of Flowering Locus T (AtFT) mRNA in both LaAG-like (type-I and -II) and LaSEP3-like (type-I), and Leafy (AtLFY) mRNAs in LaSEP3-like (type-I) transgenic plants were detected, compared to wild-type controls. Our results suggest that with controlled expression, lavender AG-like and SEP3-like genes are potentially useful for the regulation of flowering time in commercial lavender species, and could be used for plant improvement studies through molecular genetics and targeted breeding programs.
... In Arabidopsis tfl1 loss of function mutants, the inflorescence meristem ectopically expresses AP1 and LFY differentiating prematurely into a flower, and thus, the inflorescence becomes determinate (Shannon and Meeks-Wagner, 1991;Liljegren et al., 1999;Ratcliffe et al., 1999). Likewise, plants overexpressing AP1 or LFY, and even some of their downstream floral organ identity factors, like AGAMOUS (AG), also cause the conversion of the SAM into a flower (Mizukami and Ma, 1992;Mandel and Yanofsky, 1995b;Blazquez et al., 1997). The basic TFL/AP1/LFY module has been characterized in many other species and shown to be largely conserved in angiosperms, and variations in its configuration appear to correlate well with determinate/indeterminate growth habits (Pnueli et al., 1998;Liljegren et al., 1999;Ratcliffe et al., 1999;Benlloch et al., 2007;Mimida et al., 2011;Mohamed et al., 2010;Imamura et al., 2011;Iwata et al., 2012). ...
... AG is a homeotic MADS-domain transcription factor that confers carpel identity during flower development (Yanofsky et al., 1990;Bowman et al., 1991). It has been described that the constitutive expression of AG induces the differentiation of the inflorescence meristem into a terminal structure after the production of a reduced number of flowering nodes (Mizukami and Ma, 1992). As the terminal structure observed in sterile plants was mainly composed by carpel-like structures, we decided to check AG expression throughout inflorescence development in the presence (untreated wild type plants) or absence of seed development (pruned plants) by monitoring the activity of an AG::GUS reporter previously generated and characterized (Sieburth and Meyerowitz, 1997). ...
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After a vegetative phase, plants initiate the floral transition in response to both environmental and endogenous cues to optimize reproductive success. During this process, the vegetative shoot apical meristem (SAM), which was producing leaves and branches, becomes an inflorescence SAM and starts producing flowers. Inflorescences can be classified in two main categories, depending on the fate of the inflorescence meristem: determinate or indeterminate. In determinate inflorescences, the SAM differentiates directly, or after the production of a certain number of flowers, into a flower, while in indeterminate inflorescences the SAM remains indeterminate and produces continuously new flowers. Even though indeterminate inflorescences have an undifferentiated SAM, the number of flowers produced by a plant is not indefinite and is characteristic of each species, indicating that it is under genetic control. In Arabidopsis thaliana and other species with indeterminate inflorescences, the end of flower production occurs by a regulated proliferative arrest of inflorescence meristems on all reproductive branches that is reminiscent of a state of induced dormancy and does not involve the determination of the SAM. This process is controlled genetically by the FRUITFULL-APETALA2 (FUL-AP2) pathway and by a correlative control exerted by the seeds through a mechanism not well understood yet. In the absence of seeds, meristem proliferative arrest does not occur, and the SAM remains actively producing flowers until it becomes determinate, differentiating into a terminal floral structure. Here we show that the indeterminate growth habit of Arabidopsis inflorescences is a facultative condition imposed by the meristematic arrest directed by FUL and the correlative signal of seeds. The terminal differentiation of the SAM when seed production is absent correlates with the induction of AGAMOUS expression in the SAM. Moreover, terminal flower formation is strictly dependent on the activity of FUL, as it was never observed in ful mutants, regardless of the fertility of the plant or the presence/absence of the AG repression exerted by APETALA2 related factors. Seeds and FUL Control SAM Fate Balanzà et al. 2
... According to the method described by Mizukami and Ma, 1992, the scanning electron microscopy (SEM) analysis was performed. We selected the young spikelets of wild-type and mr1 at the stage of floral organ differentiation and fixed them in 3% glutaraldehyde solution at 4 • C for about 24 h. ...
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The hull (palea and lemma) is the specific organ of grass florets. Although many genes related to the hull development have been cloned, the genetic mechanisms behind the development are still unclear, and the evolutionary relationship has different explanations and heated arguments between the palea and lemma. In this study, we found a specific mr1 mutant with a reduced palea, showing an enlarged mrp and degraded bop. Phenotype observations and molecular evidences showed that the bop was converted to the mrp-like organ. Our findings first reveal that the bop and mrp are homologous structures, and the palea and lemma are the same whorl floral organs. MR1 may prevent the transformation of the bop into mrp by regulating the expressions of hull identity genes. Meantime, the mr1 mutant showed altered grain size and grain quality, with defective physical and chemical contents. MR1 was controlled by a single recessive gene and was finally located on chromosome 1, with a physical distance of 70 kb. More work will be needed for confirming the target gene of MR1, which would contribute to our understanding of grain formation and the origin between the lemma, bop, and mrp.
... In Arabidopsis thaliana, MADS-box TF gene AG is identified as the major Ctype gene. In ag mutants, the loss of AG function leads to the conversion of these organs to the perianth organs (petals and sepals) In ag mutants, the loss of AG function leads to the conversion of these organs to the perianth organs (petals and sepals) In ag mutants, the loss of AG function leads to the conversion of these organs to the perianth organs (petals and sepals).In ag mutant Arabidopsis, the loss of AG function leads to the conversion of reproductive organs to petals and sepals (Mizukami and Ma, 1992). Additionally, two genes HUA1 and HUA2, which are involved in processing of AG pre-mRNA, are also identified to have stamen and carpel identity regulatory function and hence classified under C type genes (Li et al., 2001;Cheng et al., 2003). ...
... In contrast to these reports, leaflet numbers are increased in the mtrev1-1 mutant. Previous investigations showed that REV was involved in auxin biosynthesis, transport and downstream signaling, which were tightly associated with leaf initiation and development (Huala and Sussex, 1992;Mizukami and Ma, 1992;Brandt et al., 2012). Therefore, MtREV1 probably regulates auxin-related pathways to control compound leaf formation in M. truncatula, which is evident from the opposite regulation of auxin-related genes and the content of free auxin between the mtrev1-1 mutant and 35S:MtREV1mut transformants. ...
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Leaves are derived from shoot apical meristem with three distinct dorsoventral, proximodistal and mediolateral axes. Different regulators are involved in the establishment of leaf polarity. Members of the class III homeodomain‐leucine zipper (HD‐ZIPIII) gene family are critical players in the determination of leaf adaxial identity mediated by microRNA165/166. However, their roles in compound leaf development are still unclear. By screening of a retrotransposon‐tagged mutant population of the model legume plant Medicago truncatula, a mutant line with altered leaflet numbers was isolated and characterized. Mutant leaves partially lost their adaxial identity. Leaflet numbers in the mutant were increased along the proximodistal axis, showing pinnate pentafoliata leaves in most cases, in contrast to the trifoliate leaves of wild type. Detailed characterization revealed that a lesion in a HD‐ZIPIII gene, REVOLUTA (MtREV1), resulted in the defects of the mutant. Overexpression of MtMIR166‐insensitive MtREV1 led to adaxialized leaves and ectopic leaflets along the dorsoventral axis. Accompanying the abnormal leaf patterning, the content of free auxin was affected. Our results demonstrated that MtREV1 plays a key role in determination of leaf adaxial‐abaxial polarity and compound leaf patterning, which is associated with proper auxin homeostasis. This article is protected by copyright. All rights reserved.
... Ectopic AtAG expression in Arabidopsis results in a homeotic conversion of sepals into carpel-like organs and of petals into stamens (Mizukami and Ma, 1992). Several functional studies, using AG orthologous genes from other eudicot, monocot or even gymnosperm species, showed that when ectopically expressed in Arabidopsis, they mimic the ectopic expression of AtAG, or in some cases they even rescue the ag loss of function mutants (Rutledge et al., 1998;Kitahara and Matsumoto, 2000;Zhang et al., 2004;Martin et al., 2006;Airoldi et al., 2010;Heijmans et al., 2012). ...
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Within the MADS-box gene family, the AGAMOUS-subfamily genes are particularly important for plant reproduction, because they control stamen and carpel identity. A number of studies in the last three decades have demonstrated that the AGAMOUS (AG) function has been conserved during land plant evolution. However, gene duplication events have led to subfunctionalization and neofunctionalization of AG-like genes in many species. Here we show that alternative splicing in Oryza sativa produces two variants of the AG ortholog OsMADS3 which differ in just one serine residue, S109. Interestingly, this alternative splicing variant is conserved and specific to the grass family. Since in eudicots the S109 residue is absent in AG proteins, stamen and carpel identity determination activity of the two rice isoforms was tested in Arabidopsis thaliana. These experiments revealed that only the eudicot-like OsMADS3 isoform, lacking the serine residue, had ability to specify stamens and carpels in ag mutant flowers, suggesting an important functional role for the serine residue at position 109 in AG proteins of grasses.
... FLC, FT, AG, and SEP3 were significantly higher in the ebm1 than in the wild-type "FT, " which was consistent with the results of previous studies (Schönrock et al., 2006;Schubert et al., 2006;Lopez-Vernaza et al., 2012). The AG, FT, SEP3, and FLC genes have been shown to be necessary for the early-flowering phenotype of clf mutants (Mizukami and Ma, 1992;Goodrich et al., 1997;Pelaz et al., 2001), and these target genes of CLF have antagonistic effects on the flowering time (Lopez-Vernaza et al., 2012). Of these, FT, AG, and SEP3 promote flowering, while FLC inhibits flowering in the clf mutants. ...
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Bolting is an important agronomic character of the Chinese cabbage, but premature bolting can greatly reduce its commercial value, yield, and quality. Here, early-bolting mutant 1 (ebm1) was obtained from a Chinese cabbage doubled haploid (DH) line “FT,” by using an isolated microspore culture and ethyl methanesulfonate (EMS) mutagenesis. The ebm1 was found to bolt extremely earlier than the wild type “FT.” Genetic analysis indicated that the phenotype of the ebm1 was controlled by a single recessive nuclear gene. Using a mapping population of 1,502 recessive homozygous F2 individuals with the ebm1 phenotype, the ebm1 gene was mapped to between the markers SSRhl-53 and SSRhl-61 on chromosome A04 by using SSR markers, and its physical distance was 73.4 kb. Seven genes were predicted in the target region and then cloned and sequenced; the only difference in the sequences of the ebm1 and “FT” genes was with Bra032169. Unlike that in “FT,” the Bra032169 in ebm1 had a novel 53 bp insertion that caused the termination of amino acid coding. The mutation was not consistent with EMS mutagenesis, and thus, may have been caused by spontaneous mutations during the microspore culture. Based on the gene annotation information, Bra032169 was found to encode the histone methyltransferase CURLY LEAF (CLF) in Arabidopsis thaliana. CLF regulates the expression of flowering-related genes. Further genotyping revealed that the early-bolting phenotype was fully co-segregated with the insertion mutation, suggesting that Bra032169 was the most likely candidate gene for ebm1. No significant differences were noted in the Bra032169 expression levels between the ebm1 and “FT.” However, the expression levels of the flowering-related genes FLC, FT, AG, and SEP3 were significantly higher in the ebm1 than in the “FT.” Thus, the mutation of Bra032169 is responsible for the early-bolting trait in Chinese cabbage. These results provide foundation information to help understand the molecular mechanisms of bolting in the Chinese cabbage.
... According to the ABC model, the sepal is specified by A function genes, the petal is determined by A+B function genes, the stamen is controlled by the B+C function genes, and the carpel is specified by the C class of genes (Coen and Meyerowitz, 1991;Weigel and Meyerowitz, 1994;Fourquin and Ferrandiz, 2012). In Arabidopsis, the AGAMOUS (AG) gene is the C class of gene that determines the carpel identity, specifies stamen identity with B-function genes, inhibits A-function genes and controls floral meristem determinacy (Bowman et al., 1989;Bowman et al., 1991;Mizukami and Ma, 1992;Mizukami and Ma, 1995). Subsequent studies showed that SEPALLATA (SEP) genes, expressing in four floral whorls, act as co-factors with ABC homeotic genes in specifying all types of floral organs (Theissen and Saedler, 2001;Favaro et al., 2003;Robles and Pelaz, 2005;Ruelens et al., 2017). ...
Article
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Cucumber is an important vegetable crop bearing fleshy pepo fruit harvested immature. Fruits left unpicked in time during summer production, as well as unfavorable environmental conditions during post-harvest shelf, will cause cucumber fruits to turn yellow and ripen, and thus impair the market value. Identification of maturity-related genes is of great agricultural and economic importance for cucumber production. Here, we isolated and characterized a MADS-box gene, Cucumis sativus SHATTERPROOF (CsSHP) in cucumber. Expression analysis indicated that CsSHP was specifically enriched in reproductive organs including stamens and carpels. Ectopic expression of CsSHP was unable to rescue the indehiscence silique phenotype of shp1 shp2 mutant plant in Arabidopsis. Instead, overexpression of CsSHP resulted in early flowering, precocious phenotypes, and capelloid organs in wild-type Arabidopsis. Biochemical analysis indicated that CsSHP directly interacted with cucumber SEPALLATA (SEP) proteins. CsSHP expression increased significantly during the yellowing stage of cucumber ripening, and was induced by exogenous application of abscisic acid (ABA). Therefore, CsSHP may participate in fruit maturation through the ABA pathway and floral organ specification via interaction with CsSEPs to form protein complex in cucumber.
... This suggests that the function of TFL1 in the regulation of indeterminate growth is not limited to the L1 layer. Interestingly, the MADS domain transcription factors AGAMOUS and SEPALLATA3, two important regulators of floral development, have been reported to convert the inflorescence meristem to a terminal flower when expressed from a constitutive promoter, but they had no effect on the growth habit when expression was restricted to the L1 (Mizukami and Ma, 1992;Honma and Goto, 2001;Urbanus et al., 2010). In summary, our findings indicate that TFL1 movement is crucial in the regulation of inflorescence meristem indeterminacy, although the precise region in which TFL1 activity is required remains to be determined. ...
Article
The floral transition is a critical step in the life cycle of flowering plants and several mechanisms control this finely-orchestrated process. TERMINAL FLOWER 1 (TFL1) is a floral repressor and close relative of the florigen, FLOWERING LOCUS T (FT). During the floral transition TFL1 expression is up-regulated in the inflorescence apex to maintain the indeterminate growth of the shoot apical meristem (SAM). Both TFL1 and FT are mobile proteins, but they move in different ways. FT moves from the leaves to the SAM while TFL1 appears to move within the SAM. The importance of TFL1 movement for its function in the regulation of flowering time and shoot indeterminacy and its molecular function are still largely unclear. Our results using Arabidopsis thaliana indicate that TFL1 moves from its place of expression in the centre of the SAM to the meristem layer L1 and that the movement in the SAM is required for the regulation of the floral transition. ChIP-seq and RNA-seq demonstrated that TFL1 functions as a co-transcription factor that associates with and regulates the expression of hundreds of genes. These newly identified direct TFL1 targets provide the possibility to discover new roles for TFL1 in the regulation of floral transition and inflorescence development.
... Functional studies of LLAG1, the AG homologue from lily (Benedito et al. 2004b), in Arabidopsis suggested that homeotic changes of floral organs were caused by the constitutive overexpression of LLAG1. The observed modifications were entirely in accordance with reports on AG overexpression in Arabidopsis (Mizukami and Ma 1992) and hyacinth (Li et al. 2002). These results provided additional evidence for the capability of in vivo cross-interaction of proteins belonging to the ABC model from different species, even among those which are distantly related, such as Arabidopsis and lily. ...
... In Arabidopsis, the carpels are specified by the class C gene AG alone (Fig. 7B) (Yanofsky et al. 1990, Bowman et al. 1991, Coen and Meyerowitz 1991, and overexpression of AG transforms the sepal into carpel-like organs (Mizukami and Ma 1992). Thus, AG is a key regulator in Arabidopsis, which is necessary and sufficient for carpel specification. ...
Article
The ABC model in flower development represents a milestone of plant developmental studies and is essentially conserved across a wide range of angiosperm species. Despite this overall conservation, individual genes in the ABC model are not necessarily conserved and sometimes play a species-specific role, depending on the plant. We previously reported that carpels are specified by the YABBY gene DROOPING LEAF (DL) in rice (Oryza sativa), which bears flowers that are distinct from those of eudicots. In contrast, another group reported that carpels are specified by two class C genes, OsMADS3 and OsMADS58. Here, we have addressed this controversial issue by phenotypic characterization of floral homeotic gene mutants. Analysis of a complete loss-of-function mutant of OsMADS3 and OsMADS58 revealed that carpel-like organs expressing DL were formed in the absence of the two class C genes. Furthermore, no known flower organs including carpels were specified in a double mutant of DL and SUPERWOMAN1 (a class B gene), which expresses only class C genes in whorls 3 and 4. These results suggest that, in contrast to Arabidopsis, class C genes are not a key regulator for carpel specification in rice. Instead, they seem to be involved in the elaboration of carpel morphology rather than its specification. Our phenotypic analysis also revealed that, similar to its Arabidopsis ortholog CRABS CLAW, DL plays an important function in regulating flower meristem determinacy in addition to carpel specification.
... Constitutive overexpression of LLAG1 led to homeotic changes of floral organs. The modifications observed were entirely in accordance with reports on AG over expression in Arabidopsis (Mizukami and Ma 1992) and hyacinth (Li et al. 2002). These results provided additional evidence for the capability of in vivo cross-interaction of proteins belonging to the ABC model from different species, even with those which are distantly related like Arabidopsis and lily. ...
Chapter
Flower bulbs belong to numerous botanical taxa and show remarkable diversity with regard to morphology, developmental biology, genetic control and response to the environment. In this chapter we review the current data on morpho-physiological and biochemical aspects of the transition of bulbous plants from the vegetative to the generative phase, the development of reproductive organs from initiation to anthesis, and regulation of these processes by internal and external factors. We also discuss the factors involved in florogenesis with special emphasis on the prospects and future investigations of biochemical and molecular mechanisms of florogenesis in flower bulbs. Elucidating developmental mechanisms in these species may greatly contribute to the regulation of their florogenesis, as well as to the understanding of developmental processes in other higher plants. Molecular characterization of genes involved in flower morphology will help in developing novel floral architectures in ornamental bulbs by classical breeding or by genetic manipulations using transformation systems. In addition, knowledge of bulb periodicity is essential for the control of flowering, while introducing modifications in this periodicity constitutes the basis of the techniques used to promote or delay flowering.
... In tomato, the FACIATED AND BRANCHED (FAB) receptor and the SlCLV3 (ligand) signaling pathway also control FM size [42]. In Arabidopsis, the SAM is indeterminate and the FM is determinate, while SAMs can also be determinate in many species and can vary within a single plant species [6,[43][44][45]. Precocious FM termination leads to the formation of fewer floral organs. ...
Article
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Plants, unlike animals, have developed a unique system in which they continue to form organs throughout their entire life cycle, even after embryonic development. This is possible because plants possess a small group of pluripotent stem cells in their meristems. The shoot apical meristem (SAM) plays a key role in forming all of the aerial structures of plants, including floral meristems (FMs). The FMs subsequently give rise to the floral organs containing reproductive structures. Studies in the past few decades have revealed the importance of transcription factors and secreted peptides in meristem activity using the model plant Arabidopsis thaliana. Recent advances in genomic, transcriptomic, imaging, and modeling technologies have allowed us to explore the interplay between transcription factors, secreted peptides, and plant hormones. Two different classes of plant hormones, cytokinins and auxins, and their interaction are particularly important for controlling SAM and FM development. This review focuses on the current issues surrounding the crosstalk between the hormonal and genetic regulatory network during meristem self-renewal and organogenesis.
... Constitutive overexpression of LLAG1 led to homeotic changes of floral organs. The modifications observed were entirely in accordance with reports on AG over expression in Arabidopsis (Mizukami and Ma 1992) and hyacinth (Li et al. 2002). These results provided additional evidence for the capability of in vivo cross-interaction of proteins belonging to the ABC model from different species, even with those which are distantly related like Arabidopsis and lily. ...
Chapter
Flower bulbs belong to numerous botanical taxa and show remarkable diversity with regard to morphology, developmental biology, genetic control and response to the environment. In this chapter we review the current data on morpho-physiological and biochemical aspects of the transition of bulbous plants from the vegetative to the generative phase, the development of reproductive organs from initiation to anthesis, and regulation of these processes by internal and external factors. We also discuss the factors involved in florogenesis with special emphasis on the prospects and future investigations of biochemical and molecular mechanisms of florogenesis in flower bulbs. Elucidating developmental mechanisms in these species may greatly contribute to the regulation of their florogenesis, as well as to the understanding of developmental processes in other higher plants. Molecular characterization of genes involved in flower morphology will help in developing novel floral architectures in ornamental bulbs by classical breeding or by genetic manipulations using transformation systems. In addition, knowledge of bulb periodicity is essential for the control of flowering, while introducing modifications in this periodicity constitutes the basis of the techniques used to promote or delay flowering.
... In addition, these five classes of genes are mainly MADS-box genes [12]. In Arabidopsis thaliana, APETALA1 (AP1) is a typical class A gene [13], APETALA3 (AP3) and PISTILLATA (PI) belong to the class B genes [14], and AGAMOUS (AG) is a representative gene with class C function [15]. The SEPALLATA (SEP) genes are class E genes and include SEP1, SEP2, SEP3, and SEP4 in Arabidopsis thaliana [16]. ...
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MADS-box family genes encode transcription factors that are involved in multiple developmental processes in plants, especially in floral organ specification, fruit development, and ripening. However, a comprehensive analysis of tomato MADS-box family genes, which is an important model plant to study flower fruit development and ripening, remains obscure. To gain insight into the MADS-box genes in tomato, 131 tomato MADS-box genes were identified. These genes could be divided into five groups (Mα, Mβ, Mγ, Mδ, and MIKC) and were found to be located on all 12 chromosomes. We further analyzed the phylogenetic relationships among Arabidopsis and tomato, as well as the protein motif structure and exon–intron organization, to better understand the tomato MADS-box gene family. Additionally, owing to the role of MADS-box genes in floral organ identification and fruit development, the constitutive expression patterns of MADS-box genes at different stages in tomato development were identified. We analyzed 15 tomato MADS-box genes involved in floral organ identification and five tomato MADS-box genes related to fruit development by qRT-PCR. Collectively, our study provides a comprehensive and systematic analysis of the tomato MADS-box genes and would be valuable for the further functional characterization of some important members of the MADS-box gene family.
... are also independent of the FM in petunia (Angenent and Colombo, 1996;Colombo et al., 2008). Stigmas and ovules can be formed on ectopic carpelloid structures in Arabidopsis floral development mutants (Bowman et al., 1989;Drews et al., 1991;Jack et al., 1992;Mizukami and Ma, 1992). This phenomenon can also be observed in petunia (Angenent et al., 1993(Angenent et al., , 1995. ...
... The incomplete suppression of tco-1D by ag indicates that other genes are likely misexpressed in this background as well. To test this hypothesis, we assessed the expression levels of other floral patterning and/or flowering time genes ( Figure 3F; Figure S4A-D), many of which result in leaf curling and/or early flowering when misregulated [20][21][22][23][24][25][26][27][28][29]. Numerous MADS-box floral organ identity genes were upregulated 2-fold or greater in tco-1D, including the A-class gene APETALA1 (AP1), the B-class genes AP3 and PISTILLATA (PI), the D-class genes ARABIDOPSIS B SISTER (ABS), SHATTERPROOF2 (SHP2) and SEEDSTICK (STK), and the E-class gene SEPALLATA3 (SEP3) ( Figure 3F; Figure S4A-D). ...
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As multicellular organisms grow, spatial and temporal patterns of gene expression are strictly regulated to ensure that developmental programs are invoked at appropriate stages. In this work, we describe a putative transcriptional regulator in Arabidopsis, TACO LEAF (TCO), whose overexpression results in the ectopic activation of reproductive genes during vegetative growth. Isolated as an activation-tagged allele, tco-1D displays gene misexpression and phenotypic abnormalities, such as curled leaves and early flowering, characteristic of chromatin regulatory mutants. A role for TCO in this mode of transcriptional regulation is further supported by the subnuclear accumulation patterns of TCO protein and genetic interactions between tco-1D and chromatin modifier mutants. The endogenous expression pattern of TCO and gene misregulation in tco loss-of-function mutants indicate that this factor is involved in seed development. We also demonstrate that specific serine residues of TCO protein are targeted by the ubiquitous kinase CK2. Collectively, these results identify TCO as a novel regulator of gene expression whose activity is likely influenced by phosphorylation, as is the case with many chromatin regulators.
... The MADS-box transcription factor AGAMOUS (AG) is considered to be a master regulator of floral meristem termination in Arabidopsis 3 . A loss of AG function results in indeterminate growth of floral meristems [3][4][5] . Interplay between AG and APETALA2 (AP2) regulates the expression of hundreds of targets genes 6 . ...
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In flowering plants, the switch from floral stem cell maintenance to gynoecium (female structure) formation is a critical developmental transition for reproductive success. In Arabidopsis thaliana, AGAMOUS (AG) terminates floral stem cell activities to trigger this transition. Although CRABS CLAW (CRC) is a direct target of AG, previous research has not identified any common targets. Here, we identify an auxin synthesis gene, YUCCA4 (YUC4) as a common direct target. Ectopic YUC4 expression partially rescues the indeterminate phenotype and cell wall defects that are caused by the crc mutation. The feed-forward YUC4 activation by AG and CRC directs a precise change in chromatin state for the shift from floral stem cell maintenance to gynoecium formation. We also showed that two auxin-related direct CRC targets, YUC4 and TORNADO2, cooperatively contribute to the termination of floral stem cell maintenance. This finding provides new insight into the CRC-mediated auxin homeostasis regulation for proper gynoecium formation.
... In higher plants, seeds are derived from ovules that are specified by the C, D, and E genes according to the ABCDE model (Ma and dePamphilis, 2000;Theißen et al., 2016). For instance, AGAMOUS (AG) belongs to the C class genes (Mizukami and Ma, 1992), and D class genes include STK (SEEDSTICK), SHP1 (SHATTERPROOF 1), and SHP2 in Arabidopsis (Favaro et al., 2003;Pinyopich et al., 2003). Four SEP (SEPALLATA) MADS-box genes (SEP1-4) have been identified as E class genes in Arabidopsis (Ditta et al., 2004;Pelaz et al., 2000). ...
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AGAMOUS (AG) MADS-box transcription factors have been shown to play crucial roles in floral organ and fruit development in angiosperms. Here, a tomato AG MADS-box gene, SlMBP3, was isolated. SlMBP3 is preferentially expressed in flowers and early fruit developmental stages in wild type (WT), Nr and rin mutants. Its transcripts are notably accumulated in pistils, decreased in abundance during seed and placental development and increased again during flower development. SlMBP3-RNAi tomato plants displayed fleshy placenta without jelly and extremely malformed seeds with no seed coat while SlMBP3-overexpressing plants exhibited advanced placenta liquefaction and larger seeds. Physiological feature studies showed that enzymatic activities related to cell wall modification, and the contents of cell wall components and pigments were dramatically altered in SlMBP3-RNAi placentas compared with WT. Alteration of these physiological features was also observed in SlMBP3-overexpressing placentas. Moreover, the lignin content of mature seeds in SlMBP3-RNAi lines was markedly lower than that in WT. RNA-seq and qRT-PCR analysis revealed that genes involved in seed development and the biosynthesis of cell wall modification related enzymes, gibberellin (GA), indole-3-acetic acid (Aux/IAA) and abscisic acid (ABA) were downregulated in SlMBP3-RNAi lines. These results demonstrate that SlMBP3 regulates placenta and seed development in tomato.
... Still, the original ABC model did not provide answers for some essential questions. For example, ectopic expression of ABC genes was shown to transform one type of floral organ into another, as predicted by the model, but was not sufficient to transform leaves into floral organs (Mizukami & Ma 1992;Davies et al. 1996a;Krizek & Meyerowitz 1996). These results were in conflict with an old but attractive theory of the German philosopher and writer Goethe, who proposed that all lateral organs shared a leaf-like developmental plan (Goethe 1790). ...
... The expression of AG is restricted to specific regions of stamens and carpels during flower development, and ectopic expression of AG produces an abnormal floral phenotype that resembles that of the Arabidopsis ap2 mutant (Mizukami and Ma, 1992). Our findings demonstrate that the AG 5′-UTR represses ORF translation in tobacco. ...
... In addition, the 35S::35S::CsMADS24 transgenic Arabidopsis plants displayed a homeotic transition of sepals into carpelloid-like structures with earlier appearance of stigmatic papillae, suggesting that ectopic expression of CsMADS24 is sufficient to convert sepals into carpels, but insufficient to convert petals into stamens. These results indicate that 35S::35S::CsMADS24 transgenic Arabidopsis plants only had partially similar phenotypes compared to the Arabidopsis plants with AG ectopic expression [41]. Similar phenotypes were also reported previously for the ectopic expression of AG orthologs from several plant species. ...
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The floral homeotic C-function MADS gene AGAMOUS (AG) in Arabidopsis plays crucial roles in specifying stamen and carpel identities as well as determining floral meristem. However, there have been only a few studies of floral homeotic C-function genes in cucumber thus far. In the present study, CsMADS24, a putative AG ortholog from cucumber, was isolated and characterized. Sequence analysis and protein sequence alignment revealed that the deduced CsMADS24 protein contained the typical MIKC structure and the N-terminal extension, as well as two highly conserved AG motifs (I and II). Phylogenetic analysis showed that CsMADS24 fell into the clade of core eudicots, while being distant from the AG orthologs of basal eudicots, monocots and gymnosperms. Expression analysis by RT-PCR showed that CsMADS24 was exclusively expressed in female flower buds. In situ hybridization revealed that CsMADS24 expression was only detected in the carpels. Functional analyses indicated that the sepals were partly converted into carpelloid-like structures in 35S::35S::CsMADS24 transgenic plants. In addition, earlier flowering and delayed floral organ abscission during the development of siliques were also observed in transgenic Arabidopsis. Our findings demonstrate that the AG ortholog plays an exclusive role in carpel specification of cucumber, providing a basis for revealing the mechanisms of reproductive development in cucumber.
... The study revealed that critical regulatory genes for spikelet meristem development might be conserved between Phyllostachys and Oryza. For example, genes from the AG-like subfamily are essential for reproductive structure morphogenesis [69][70][71]. As shown in Fig. 6, bamboo AG-like genes PeMADS1, − 29, and − 31 are primarily expressed in the later stages of floral development, whereas their Oryza orthologs OsMADS3 and − 58 have relatively greater expression levels in reproductive tissues [18]. ...
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Background: MADS-box genes encode a large family of transcription factors that play significant roles in plant growth and development. Bamboo is an important non-timber forest product worldwide, but previous studies on the moso bamboo (Phyllostachys edulis) MADS-box gene family were not accurate nor sufficiently detailed. Results: Here, a complete genome-wide identification and characterization of the MADS-box genes in moso bamboo was conducted. There was an unusual lack of type-I MADS-box genes in the bamboo genome database ( http://202.127.18.221/bamboo/index.php ), and some of the PeMADS sequences are fragmented and/or inaccurate. We performed several bioinformatics techniques to obtain more precise sequences using transcriptome assembly. In total, 42 MADS-box genes, including six new type-I MADS-box genes, were identified in bamboo, and their structures, phylogenetic relationships, predicted conserved motifs and promoter cis-elements were systematically investigated. An expression analysis of the bamboo MADS-box genes in floral organs and leaves revealed that several key members are involved in bamboo inflorescence development, like their orthologous genes in Oryza. The ectopic overexpression of one MADS-box gene, PeMADS5, in Arabidopsis triggered an earlier flowering time and the development of an aberrant flower phenotype, suggesting that PeMADS5 acts as a floral activator and is involved in bamboo flowering. Conclusion: We produced the most comprehensive information on MADS-box genes in moso bamboo. Additionally, a critical PeMADS gene (PeMADS5) responsible for the transition from vegetative to reproductive growth was identified and shown to be related to bamboo floral development.
... The timing and location of AG expression are critical for cell fate determination during flower development. During early flower development, AG is specifically expressed in the region of the floral meristem that gives rise to stamens and carpels [26, [71][72][73][74][75]. Misexpression of AG leads to ectopic formation of reproductive tissues, whereas partially reduced AG expression only affects determinacy [26, 76,77]. The existence of many tissuespecific transcription factors for regulating AG expression supports the importance of a proper spatial distribution of AG mRNA in the floral meristem for establishing robust tissue patterning (Table 1). ...
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Tissue-specific transcription factors allow cells to specify new fates by exerting control over gene regulatory networks and the epigenetic landscape of a cell. However, our knowledge of the molecular mechanisms underlying cell fate decisions is limited. In Arabidopsis, the MADS-box transcription factor AGAMOUS (AG) plays a central role in regulating reproductive organ identity and meristem determinacy during flower development. During the vegetative phase, AG transcription is repressed by Polycomb complexes and intronic noncoding RNA. Once AG is transcribed in a spatiotemporally regulated manner during the reproductive phase, AG functions with chromatin regulators to change the chromatin structure at key target gene loci. The concerted actions of AG and the transcription factors functioning downstream of AG recruit general transcription machinery for proper cell fate decision. In this review, we describe progress in AG research that has provided important insights into the regulatory and epigenetic mechanisms underlying cell fate determination in plants.
... This process repeats itself indeterminately, resulting in the flower-inflower phenotype [9,10,13]. Constitutive expression of AG results in carpeloid sepals and staminoid petals in the first and second whorl [14]. It is known that AG activity inhibits AP2 function and, vice versa, AP2 function represses AG activity [15]. ...
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Gynoecium development is dependent on gene regulation and hormonal pathway interactions. The phytohormones auxin and cytokinin are involved in many developmental programs, where cytokinin is normally important for cell division and meristem activity, while auxin induces cell differentiation and organ initiation in the shoot. The MADS-box transcription factor AGAMOUS (AG) is important for the development of the reproductive structures of the flower. Here, we focus on the relationship between AG and cytokinin in Arabidopsis thaliana, and use the weak ag-12 and the strong ag-1 allele. We found that cytokinin induces carpeloid features in an AG-dependent manner and the expression of the transcription factors CRC, SHP2, and SPT that are involved in carpel development. AG is important for gynoecium development, and contributes to regulating, or else directly regulates CRC, SHP2, and SPT. All four genes respond to either reduced or induced cytokinin signaling and have the potential to be regulated by cytokinin via the type-B ARR proteins. We generated a model of a gene regulatory network, where cytokinin signaling is mainly upstream and in parallel with AG activity.
... The model thus states that the formation of sepals requires the activities of class A and E genes; petals require A, B and E activities; stamens require B, C and E activities; and carpels require C and E activities (Theissen et al., 2000). The model also confers a cadastral function to class A and C genes: class A and C genes repress each other within their functional domains (Fig 20-B, (Drews et al., 1991;Mizukami and Ma, 1992). ...
Thesis
In plants, the development of aerial organs is indeterminate: it takes place throughout their lifespan. In contrast, the development of floral organs is determinate in Arabidopsis thaliana, each flower has the same number of floral organs. This difference in development is due to the maintenance or not of the pool of stem cells present in the stem cell niches, the meristems. During my thesis I showed that the transcriptional regulator VIP3 contributes to the regulation of the switch from indeterminate to determinate in flowers. This also revealed that the control of flower termination is not as robust as classically thought. Because VIP3 is also involved in the regulation of epigenetic marks and response to external mechanical stimuli, this work opens new questions on the role of mechanical signals in indeterminacy. On a more technical standpoint, the analysis of shoot development suffers from a lack of imaging methods with high temporal resolution and in-depth optical sectioning. During the last decade, light sheet microscopy has emerged as a competitive imaging modality in developmental biology. However, in plants, the technique has mainly been used in roots because of limits in the microscope design. During my thesis, I developed protocols allowing the imaging of aerial organs in A. thaliana using a novel light sheet set-up (Phaseview Alpha3) where shoot samples can be observed while in water. I set up an imaging pipeline from sample mounting to quantitative analysis, with a focus on local dynamics of microtubules in cotyledon epidermis in relation to cell shape. Altogether, this work provides both conceptual and technical prospects for future quantitative projects in plant development.
... Some of these WOX genes (including WUSHEL) regulate ovule development, floral organogenesis, floral transition, and participate in gynoecium and embryo development [67,68]. In Arabidopsis, WUSCHEL also activates the AGAMOUS (AG) gene, a class C gene required for normal development of carpels in flowers [69][70][71]. Other WOX genes in Arabidopsis are also capable to alter the expression of the AGAMOUS gene [72]. ...
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Background: Carica papaya is a trioecious plant species with a genetic sex-determination system defined by sex chromosomes. Under unfavorable environmental conditions male and hermaphrodite exhibit sex-reversal. Previous genomic research revealed few candidate genes for sex differentiation in this species. Nevertheless, more analysis is still needed to identify the mechanism responsible for sex flower organ development in papaya. Results: The aim of this study was to identify differentially expressed genes among male, female and hermaphrodite flowers in papaya during early (pre-meiosis) and later (post-meiosis) stages of flower development. RNA-seq was used to evaluate the expression of differentially expressed genes and RT-qPCR was used to verify the results. Putative functions of these genes were analyzed based on their homology with orthologs in other plant species and their expression patterns. We identified a Male Sterility 1 gene (CpMS1) highly up-regulated in male and hermaphrodite flower buds compared to female flower buds, which expresses in small male flower buds (3-8 mm), and that might be playing an important role in male flower organ development due to its homology to MS1 genes previously identified in other plants. This is the first study in which the sex-biased expression of genes related to tapetum development in the anther developmental pathway is being reported in papaya. Besides important transcription factors related to flower organ development and flowering time regulation, we identified differential expression of genes that are known to participate in ABA, ROS and auxin signaling pathways (ABA-8-hydroxylases, AIL5, UPBEAT 1, VAN3-binding protein). Conclusions: CpMS1 was expressed in papaya male and hermaphrodite flowers at early stages, suggesting that this gene might participate in male flower organ development processes, nevertheless, this gene cannot be considered a sex-determination gene. Due to its homology with other plant MS1 proteins and its expression pattern, we hypothesize that this gene participates in anther development processes, like tapetum and pollen development, downstream gender specification. Further gene functional characterization studies in papaya are required to confirm this hypothesis. The role of ABA and ROS signaling pathways in papaya flower development needs to be further explored as well.
Article
AGAMOUS/SEEDSTICK (AG/STK) subfamily genes play crucial roles in the reproductive development of plants. However, most of our current knowledge of AG/STK subfamily genes is restricted to core eudicots and grasses, and the knowledge of ancestral exon-intron structures, expression patterns, protein-protein interaction patterns, and functions of AG/STK subfamily genes remains unclear. To determine these, we isolated AG/STK subfamily genes (MawuAG1, MawuAG2 and MawuSTK) from a woody basal angiosperm Magnolia wufengensis. MawuSTK arose from the gene duplication event occurring before the diversification of extant angiosperms, and MawuAG1 and MawuAG2 may result from a gene duplication event occurring before the divergence of Magnoliaceae and Lauraceae. Gene duplication led to apparent diversification in their expression and interaction patterns. It revealed that expression in both stamens and carpels likely represents the ancestral expression profiles of AG lineage genes, and expression of STK-like genes in stamens may have been lost soon after the appearance of STK lineage. Moreover, AG/STK subfamily proteins may have immediately established interactions with the SEPALLATA (SEP) subfamily proteins following the emergence of SEP subfamily; however, their interactions with the APETALA1/FRUITFULL subfamily proteins or themselves, differ from those found in monocots, and basal and core eudicots. MawuAG1 plays highly conserved roles in the determinacy of stamen, carpel and ovule identity, while gene duplication contributed to the functional diversification of MawuAG2 and MawuSTK. In addition, we investigated the evolutionary history of exon-intron structural changes of AG/STK subfamily, and a novel splice-acceptor mode (GUU-AU) and the convergent evolution of N-terminal extension in the euAG and PLE subclades were revealed for the first time. These results further advance our understanding of ancestral AG/STK subfamily genes in terms of phylogeny, exon-intron structures, expression and interaction patterns, and functions, and provide strong evidences in the significance for gene duplication in the expansion and evolution of the AG/STK subfamily.
Thesis
Les plantes conservent la capacité à former de nouveaux organes tout au long de leur vie grâce au maintien de structures contenant des cellules souches, les méristèmes. La formation des fleurs, structures reproductives de la plante, est une étape essentielle de son cycle de vie. Afin d’assurer un développement floral complet, un méristème doit être formé de novo au sein du jeune bouton floral. Des données éparses de la littérature indiquent que le facteur de transcription LEAFY, en plus d’être un régulateur clé de l’identité florale, est aussi impliqué dans la mise en place du méristème floral.Dans la première partie de ce travail nous explorons le rôle de LEAFY dans l’initiation du méristème floral. Cette étude est concentrée sur un gène cible de LEAFY, le facteur de transcription REGULATOR OF AXILLARY MERISTEMS1 (RAX1). Nous montrons notamment que la voie régulée par LEAFY/RAX1 agit en parallèle du facteur de transcription REVOLUTA pour permettre la mise en place du méristème floral.Dans la deuxième partie de ce travail nous étudions les propriétés du domaine N-terminal de LEAFY. Ce domaine permet l’oligomérisation de LEAFY ainsi que potentiellement sa liaison aux régions fermées de la chromatine. Nous étudions également de manière plus exploratoire le rôle de ce domaine dans la régulation de l’expression du gène AGAMOUS, un important régulateur du développement floral.
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Litsea cubeba (Lour.) Pers. (mountain pepper, Lauraceae) is an important woody essential oil crop that produces fragrant oils in its fruits, especially in its peels. Identification of genes involved in the regulation of fruits and peel architecture is of economic significance for L. cubeba industry. It has been well known that the MADS-box genes are essential transcription factors that control flowers and fruits development. Here, we obtained 33 MADS-box genes firstly from the RNA-seq data in L. cubeba, and 27 of these genes were of the MIKC-type. LcMADS20, an AGAMOUS-like gene, was highly expressed in the developing stages of fruits, particularly at 85 days after full bloom. The ectopic expression of LcMADS20 in Arabidopsis resulted in not only curved leaves, early flowering and early full-opened inflorescences, but also shorter siliques and decreased percentage of peel thickness. Moreover, in the LcMADS20 transgenic Arabidopsis, the expression modes of several intrinsic ABC model class genes were influenced, among which the expression of FUL was significantly reduced and AP3, AG, and STK were significantly increased. This study systematically analyzed the MADS-box genes in L. cubeba at the transcriptional level and showed that LcMADS20 plays important roles in the regulation of fruit architecture.
Article
In order to ascertain the regulatory mechanism of fruit development in Isatis indigotica Fortune, the complementary DNA (cDNA) sequence of the SHATTERPROOF 2 (SHP2) orthologous gene was identified by Rapid Amplification of cDNA Ends technology and the corresponding gene was named IiSHP2. The expression pattern of IiSHP2 was determined by quantitative reverse transcription-polymerase chain reaction and wild-type Col-0 Arabidopsis plants were transformed with the IiSHP2 gene using Agrobacterium tumefaciens and the floral-dip method. Expression analyses indicated that IiSHP2 was highly expressed in flowers, silicles and seeds. Compared to wild-type plants, IiSHP2 transgenic lines bolted earlier. Detailed phenotypic observations showed that the size of the rosette and cauline leaves in transgenic lines was reduced and the cauline leaves of the transgenic lines were incurved and displayed a funnel-like shape. During the reproductive growth stage, IiSHP2 transgenic plants produced shortened sepals and the flower buds were not encapsulated completely. Moreover, the petals of the transgenic lines were converted into stamineous tissues, accompanied by exposed stamens, short malformed siliques and wrinkled valves, indicating a severe decline in fertility. These experimental conclusions are valuable as a reference for the breeding of medicinal plants.
Chapter
The development of flowers and floral organs is directed by intricate genetic programmes, many aspects of which appear to be shared among angiosperms. Early acting genes establish floral meristem identity in flower primordia initiated at the periphery of the inflorescence meristem. Later, floral organ primordia arise at precise positions within these floral meristems and take on one of the four distinct identities (sepals, petals, stamens and carpels). The ABCE model, supported by both molecular and genetic experiments in Arabidopsis , explains how a small number of regulatory genes (called floral homeotic genes or floral organ identity genes) act in different combinations to specify these different organ types. The floral organ identity genes encode transcription factors that form distinct higher order protein complexes in different regions of a flower primordium to control the expression of target genes responsible for organogenesis. Key Concepts • Lateral organs produced by the shoot apical meristem during reproductive development acquire their identity as flowers through the action of floral meristem identity genes such as LEAFY and APETALA1 . • The identities of each of the four organ types of a flower (sepal, petal, stamen and carpel) are conferred by a unique combination of floral organ identity gene activities, referred to as classes A, B, C and E in the ABCE model. • The activities of the class A, B and C genes are restricted to particular regions within a developing flower primarily, but not exclusively, through transcriptional regulation. • The MADS domain transcription factors encoded by the class A, B, C and E genes form unique tetrameric transcriptional regulatory complexes in cells of each floral whorl. • The transcriptional regulatory complexes formed by the A, B, C and E proteins regulate distinct sets of genes at different stages of flower development. • Many aspects of the genetic programmes conferring floral meristem identity and floral organ identity are conserved among all angiosperms.
Article
E-class MADS-box genes, SEPALLATA (SEP), participate in various aspects of plant development together with B-, C- and D-class MADS-box genes. IiSEP4, a homologous gene of SEP4, was cloned from Isatis indigotica. IiSEP4 was highly expressed in sepals, and its mRNA was mildly detected in leaves, inflorescences, flowers, stamens and young silicles. Constitutive expression of IiSEP4 in Arabidopsis thaliana caused early flowering, accompanied by the reduction of flowers and floral organs. Moreover, the sepals in some flowers were transformed into carpelloid structures with stigmatic papillae, and obviously accompanied by ovule formation. Yeast two-hybrid assays demonstrated that IiSEP4 interacts with other woad MADS proteins to determine the identity of floral organs. These findings reveal the important roles of IiSEP4 in floral development of I. indigotica. The results of this study can lay a foundation for further study on biological functions of MADS transcription factors in I. indigotica.
Article
Vitis vinifera L. can be divided into two subspecies, V. vinifera subsp. vinifera, the cultivated grapevine, and its wild ancestor, V. vinifera subsp. sylvestris. Three flower types have been described: hermaphrodite and female in some varieties of vinifera, and male or female flowers in sylvestris. We have conducted an expression analysis of the functional genes candidate to sex determination in the newly defined sex locus described by Picq et al (2014) using four flower types. The candidate gene Ethylene overproducer-1 (ETO1) localized in the sex locus region and which inhibits the enzyme activity of the enzyme ACS (1-aminocyclopropane-1-carboxylic acid synthase) was showed highly significantly different expression pattern according to the sex flower. Other genes studied in the sex locus do not reveal significant different expression patterns. For genes located outside of the sex locus, only the SAUR (Small auxin up RNAs) protein and the ACS gene showed different expression among sex flowers. Therefore, as ETO1 is only expressed in female and hermaphrodite flowers, it could be a good candidate for the recessive female fertility mutation and ACS copy could be implied in the reaction cascade leading to the inhibition of stamens in female flowers. However, the ETO1 only negatively interacts with type 2 ACS and our ACS phylogeny analysis confirmed that the VviACS copy is not type 2. Therefore, it is unlikely that there is such molecular interaction in grapevine. Another hypothesis could be that the molecular mechanisms that regulated the activity of VviACS2 are induced by the VvETO1 protein regulating the activity of both families of ACS type I and type 2. The last gene showing differential expression according to sex is the SAUR protein. This gene consists in early auxin response genes family playing key role in hormonal and environmental signals. Our results pointed out that one gene (ETO1) inside of the flower sex locus region and two genes (ACS, SAUR) located outside of the sex locus region, could be considered as putative candidate genes for the control of sexual traits in grapevine.
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The link between gene regulation and morphogenesis of multicellular organisms is a fundamental problem in biology. We address this question in the floral meristem of Arabidopsis, which generates new tissues and organs through complex changes in growth patterns. Starting from high-resolution time-lapse images, we generated a comprehensive 4-D atlas of early flower development including cell lineage, cellular growth rates and the expression patterns of 28 regulatory genes. This information was introduced in MorphoNet, a web-based open-access platform. The application of mechanistic computational models indicated that the molecular network based on the literature only explained a minority of the expression patterns. This was substantially improved by adding single regulatory hypotheses for individual genes. We next used the integrated information to correlate growth with the combinatorial expression of multiple genes. This led us to propose a set of hypotheses for the action of individual genes in morphogenesis, not visible by simply correlating gene expression and growth. This identified the central transcription factor LEAFY as a potential regulator of heterogeneous growth, which was supported by quantifying growth patterns in a leafy mutant. By providing an integrated, multiscale view of flower development, this atlas should represent a fundamental step towards mechanistic multiscale-scale models of flower development.
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Unlike the flower of the model monocot rice, which has diverged greatly from the ancestral monocot flower, the pineapple ( Ananas comosus ) flower is more typical of monocot flowers. Here, we identified 43 pineapple genes containing MADS-box domains, including 11 type I and 32 type II genes. RNA-seq expression data generated from five pineapple floral organs (sepals, petals, stamens, pistils, and ovules) and quantitative real-time PCR revealed tissue-specific expression patterns for some genes. We found that AcAGL6 and AcFUL1 were mainly expressed in sepals and petals, suggesting their involvement in the regulation of these floral organs. A pineapple ‘ABCDE’ model was proposed based on the phylogenetic analysis and expression patterns of MADS-box genes. Unlike rice and orchid with frequent species-specific gene duplication and subsequent expression divergence, the composition and expression of the ABCDE genes were conserved in pineapple. We also found that AcSEP1/3 , AcAG , AcAGL11a/b/c , and AcFUL1 were highly expressed at different stages of fruit development and have similar expression profiles, implicating these genes’ role in fruit development and ripening processes. We propose that the pineapple flower can be used as a model for studying the ancestral form of monocot flowers to investigate their development and evolutionary history.
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“From our acquaintance with this abnormal enabled to unveil the secrets that normal us, and to see distinctly what, from the regular we can only infer.” - J. W. von Goethe (1790)
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Growth of flowering stems in wild-type Arabidopsis is indeterminate. Many flowers arise sequentially on the flanks of apical meristems in a phyllotactic spiral. We have isolated eight recessive mutants of a gene, terminal flower, in which inflorescences become determinate. Flower primordia sooner or later ‘invade’ the meristem summit leading to cessation of its further growth. Primary apical meristems usually terminate with several part-flowers which lack pedicels, and several normal pedicellate flowers may arise first. By contrast apical meristems of secondary branches usually produce only a single pedicellate flower. Plant height is also reduced and more rosette inflorescences develop. These growth patterns occur in six strong mutants raised at 25°C under continuous light. In two weak mutants termination occurs much later with many more flowers arising before eventual termination. Termination is similarly delayed in at least one of the strong mutants grown at lower temperatures. The tfl mutation does not affect the indeterminate growth of flower meristems, at least in-so-far as this occurs in agamous mutants. The tfl locus is at the top of linkage group 5, close to RFLP 447. We propose that the TFL gene product supports the activity of an inhibitor of flower primordium initiation. This inhibitor would normally prevent flowers from arising on the inflorescence apex but in tfl mutants it may readily fall below its threshold of activity. The TFL gene may be one of a class responsible for evolutionary changes between indeterminate and determinate growth.
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The first step in flower development is the generation of a floral meristem by the inflorescence meristem. We have analyzed how this process is affected by mutant alleles of the Arabidopsis gene LEAFY. We show that LEAFY interacts with another floral control gene, APETALA1, to promote the transition from inflorescence to floral meristem. We have cloned the LEAFY gene, and, consistent with the mutant phenotype, we find that LEAFY RNA is expressed strongly in young flower primordia. LEAFY expression procedes expression of the homeotic genes AGAMOUS and APETALA3, which specify organ identify within the flower. Furthermore, we demonstrate that LEAFY is the Arabidopsis homolog of the FLORICAULA gene, which controls floral meristem identity in the distantly related species Antirrhinum majus.
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The predicted products of floral homeotic genes, AGAMOUS (AG) from Arabidopsis thaliana and DEFICIENS A (DEF A) from Antirrhinum majus, have been shown previously to share strong sequence similarity with transcription factors from humans (SRF) and yeast (MCM1). The conserved sequence between these proteins is localized within a domain known to be necessary for the DNA binding and for the dimerization of SRF. We have isolated six new genes from A. thaliana, AGL1-AGL6, which also have this conserved sequence motif. On the basis of the sequence comparison between the AG and AGL genes, they can be assigned to two subfamilies of a large gene family. RNA dot blot analysis indicates that five of these genes (AGL1, AGL2, AGL4, AGL5, and AGL6) are preferentially expressed in flowers. In addition, in situ RNA hybridization experiments with AGL1 and AGL2 show that their mRNAs are detected in some floral organs but not in others. Our results suggest that these genes may act to control many steps of Arabidopsis floral morphogenesis. In contrast, the AGL3 gene is expressed in vegetative tissues as well as in flowers, suggesting that it functions in a broader range of tissues. We discuss possible roles of this gene family during the evolution of flowers.
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We describe allelic series for three loci, mutations in which result in homeotic conversions in two adjacent whorls in the Arabidopsis thaliana flower. Both the structure of the mature flower and its development from the initial primordium are described by scanning electron microscopy. New mutations at the APETALA2 locus, ap2-2, ap2-8 and ap2-9, cause homeotic conversions in the outer two whorls: sepals to carpels (or leaves) and petals to stamens. Two new mutations of PISTILLATA, pi-2 and pi-3, cause second and third whorl organs to differentiate incorrectly. Homeotic conversions are petals to sepals and stamens to carpels, a pattern similar to that previously described for the apetala3-1 mutation. The AGAMOUS mutations, ag-2 and ag-3, affect the third and fourth whorls and cause petals to develop instead of stamens and another flower to arise in place of the gynoecium. In addition to homeotic changes, mutations at the APETALA2, APETALA3 and PISTILLATA loci may lead to reduced numbers of organs, or even their absence, in specific whorls. The bud and flower phenotypes of doubly and triply mutant strains, constructed with these and previously described alleles, are also described. Based on these results, a model is proposed that suggests that the products of these homeotic genes are each active in fields occupying two adjacent whorls, AP2 in the two outer whorls, PI and AP3 in whorls two and three, and AG in the two inner whorls. In combination, therefore, the gene products in these three concentric, overlapping fields specify the four types of organs in the wild-type flower. Further, the phenotypes of multiple mutant lines indicate that the wild-type products of the AGAMOUS and APETALA2 genes interact antagonistically. AP2 seems to keep the AG gene inactive in the two outer whorls while the converse is likely in the two inner whorls. This field model successfully predicts the phenotypes of all the singly, doubly and triply mutant flowers described.
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The analysis of mutations affecting flower structure has led to the identification of some of the genes that direct flower development. Cloning of these genes has allowed the formulation of molecular models of how floral meristem and organ identity may be specified, and has shown that the distantly related flowering plants Arabidopsis thaliana and Antirrhinum majus use homologous mechanisms in floral pattern formation.
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Mutations in the AGAMOUS (AG) gene cause transformations in two adjacent whorls of the Arabidopsis flower. Petals develop in the third floral whorl rather than the normal stamens, and the cells that would normally develop into the fourth whorl gynoecium behave as if they constituted an ag flower primordium. Early in flower development, AG RNA is evenly distributed throughout third and fourth whorl organ primordia but is not present in the organ primordia of whorls one and two. In contrast to the early expression pattern, later in flower development, AG RNA is restricted to specific cell types within the stamens and carpels as cellular differentiation occurs in those organs. Ectopic AG expression patterns in flowers mutant for the floral homeotic gene APETELA2 (AP2), which regulates early AG expression, suggest that the late AG expression is not directly dependent on AP2 activity.
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Cells in developing organisms do not only differentiate, they differentiate in defined patterns. A striking example is the differentiation of flowers, which in most plant families consist of four types of organs: sepals, petals, stamens and carpels, each composed of characteristic cell types. In the families of flowering plants in which these organs occur, they are patterned with the sepals in the outermost whorl or whorls of the flower, with the petals next closest to the center, the stamens even closer to the center, and the carpels central. In each species of flowering plant the disposition and number (or range of numbers) of these organs is also specified, and the floral 'formula' is repeated in each of the flowers on each individual plant of the species. We do not know how cells in developing plants determine their position, and in response to this determination differentiate to the cell types appropriate for that position. While there have been a number of speculative proposals for the mechanism of organ specification in flowers (Goethe, 1790; Goebel, 1900; Heslop-Harrison, 1964; Green, 1988), recent genetic evidence is inconsistent with all of them, at least in the forms in which they were originally presented (Bowman et al. 1989; Meyerowitz et al. 1989). We describe here a preliminary model, based on experiments with Arabidopsis thaliana. The model is by and large consistent with existing evidence, and has predicted the results of a number of genetic and molecular experiments that have been recently performed.
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Deficiens (defA+) is a homeotic gene involved in the genetic control of Antirrhinum majus flower development. Mutation of this gene (defA-1) causes homeotic transformation of petals into sepals and of stamina into carpels in flowers displaying the 'globifera' phenotype, as shown by cross sections and scanning electronmicroscopy of developing flowers. A cDNA derived from the wild type defA+ gene has been cloned by differential screening of a subtracted 'flower specific' cDNA library. The identity of this cDNA with the defA+ gene product has been confirmed by utilizing the somatic and germinal instability of defA-1 mutants. According to Northern blot analyses the defA+ gene is expressed in flowers but not in leaves, and its expression is nearly constant during all stages of flower development. The 1.1 kb long mRNA has a 681 bp long open reading frame that can code for a putative protein of 227 amino acids (mol. wt 26.2 kd). At its N-terminus the DEF A protein reveals homology to a conserved domain of the regulatory proteins SRF (activating c-fos) in mammals and GRM/PRTF (regulating mating type) in yeast. We discuss the structure and the possible function of the DEF A protein in the control of floral organogenesis.
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Mutations in the homeotic gene agamous of the plant Arabidopsis cause the transformation of the floral sex organs. Cloning and sequence analysis of agamous suggest that it encodes a protein with a high degree of sequence similarity to the DNA-binding region of transcription factors from yeast and humans and to the product of a homeotic gene from Antirrhinum. The agamous gene therefore probably encodes a transcription factor that regulates genes determining stamen and carpel development in wild-type flowers.
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We have characterized the floral phenotypes produced by the recessive homeotic apetala 1-1 (ap1-1) mutation in Arabidopsis. Plants homozygous for this mutation display a homeotic conversion of sepsis into brachts and the concomitant formation of floral buds in the axil of each transformed sepal. In addition, these flowers lack petals. We show that the loss of petal phenotype is due to the failure of petal primordia to be initiated. We have also constructed double mutant combinations with ap1 and other mutations affecting floral development. Based on these results, we suggest that the AP1 and the apetala 2 (AP2) genes may encode similar functions that are required to define the pattern of where floral organs arise, as well as for determinate development of the floral meristem. We propose that the AP1 and AP2 gene products act in concert with the product of the agamous (AG) locus to establish a determinate floral meristem, whereas other homeotic gene products are required for cells to differentiate correctly according to their position. These results extend the proposed role of the homeotic genes in floral development and suggest new models for the establishment of floral pattern.
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The early development of the flower of Arabidopsis thaliana is described from initiation until the opening of the bud. The morphogenesis, growth rate, and surface structure of floral organs were recorded in detail using scanning electron microscopy. Flower development has been divided into 12 stages using a series of landmark events. Stage 1 begins with the initiation of a floral buttress on the flank of the apical meristem. Stage 2 commences when the flower primordium becomes separate from the meristem. Sepal primordia then arise (stage 3) and grow to overlie the primordium (stage 4). Petal and stamen primordia appear next (stage 5) and are soon enclosed by the sepals (stage 6). During stage 6, petal primordia grow slowly, whereas stamen primordia enlarge more rapidly. Stage 7 begins when the medial stamens become stalked. These soon develop locules (stage 8). A long stage 9 then commences with the petal primordia becoming stalked. During this stage all organs lengthen rapidly. This includes the gynoecium, which commences growth as an open-ended tube during stage 6. When the petals reach the length of the lateral stamens, stage 10 begins. Stigmatic papillae appear soon after (stage 11), and the petals rapidly reach the height of the medial stamens (stage 12). This final stage ends when the 1-millimeter-long bud opens. Under our growing conditions 1.9 buds were initiated per day on average, and they took 13.25 days to progress through the 12 stages from initiation until opening.
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We describe the effects of four recessive homeotic mutations that specifically disrupt the development of flowers in Arabidopsis thaliana. Each of the recessive mutations affects the outcome of organ development, but not the location of organ primordia. Homeotic transformations observed are as follows. In agamous-1, stamens to petals; in apetala2-1, sepals to leaves and petals to staminoid petals; in apetala3-1, petals to sepals and stamens to carpels; in pistillata-1, petals to sepals. In addition, two of these mutations (ap2-1 and pi-1) result in loss of organs, and ag-1 causes the cells that would ordinarily form the gynoecium to differentiate as a flower. Two of the mutations are temperature-sensitive. Temperature shift experiments indicate that the wild-type AP2 gene product acts at the time of primordium initiation; the AP3 product is active later. It seems that the wild-type alleles of these four genes allow cells to determine their place in the developing flower and thus to differentiate appropriately. We propose that these genes may be involved in setting up or responding to concentric, overlapping fields within the flower primordium.
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Variation in plant shoot structure may be described as occurring through changes within a basic unit, the metamer. Using this terminology, the apical meristem of Arabidopsis produces three metameric types sequentially: type 1, rosette; type 2, coflorescence-bearing with bract; and type 3, flower-bearing without bract. We describe a mutant of Arabidopsis, Leafy, homozygous for a recessive allele of a nuclear gene LEAFY (LFY), that has an inflorescence composed only of type 2-like metamers. These data suggest that the LFY gene is required for the development of type 3 metamers and that the transition from type 2 to type 3 metamers is a developmental step distinct from that between vegetative and reproductive growth (type 1 to type 2 metamers). Results from double mutant analysis, showing that lfy-1 is epistatic to the floral organ homeotic gene ap2-6, are consistent with the hypothesis that a functional LFY gene is necessary for the expression of downstream genes controlling floral organ identity.
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We describe a novel mutant of Arabidopsis, Flo10, which is the result of a recessive allele, flo10, in the nuclear gene FLO10. The first three organ whorls (sepals, petals, and stamens) of Flo10 flowers are normal, but the fourth, gynoecial whorl is replaced by two to eight stamens or stamen-carpel intermediate organs. Studies of ontogeny suggest that the position of the first six of these fourth-whorl organs often resembles that of the wild-type third-whorl organs. To determine the interaction of the FLO10 gene with the floral organ homeotic genes APETALA2 (AP2), PISTILLATA (PI), AP3, and AGAMOUS (AG), we generated lines homozygous for flo10 and heterozygous or homozygous for a recessive allele of the homeotic genes. On the basis of our data, we suggest that FLO10 functions to prevent the expression of the AP3/PI developmental pathway in the gynoecial (fourth) whorl.
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We have examined the floral morphology and ontogeny of three mutants of Arabidopsis thaliana, Ap2-5, Ap2-6, and Ap2-7, that exhibit homeotic changes of the perianth organs because of single recessive mutations in the AP2 gene. Homeotic conversions observed are: sepals to carpels in all three mutants, petals to stamens in Ap2-5, and petals to carpels in Ap2-6. Our analysis of these mutants suggests that the AP2 gene is required early in floral development to direct primordia of the first and second whorls to develop as perianth rather than as reproductive organs. In addition, our results support one of the two conflicting hypotheses concerning the structures of the calyx and the gynoecium in the Brassicaceae.
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We have isolated a number of mutants of Arabidopsis thaliana, a member of the mustard family, that have defects in flower development and morphogenesis. Of these, five mutants have been extensively characterized. Two mutants (Fl-40, Fl-48) lacking petals show homeotic conversion of sepals to carpels. One mutant (Fl-54) displays highly variable phenotypes, including several types of homeotic variations, loss or distored positions of the floral organs as well as abnormal structures on the inflorescence. Two other mutants (Fl-82, Fl-89) show aberrant structures in the pistils. Genetic analyses have revealed that these mutations are single and recessive, except for one mutant whose mutational loci still remain to be determined. These mutants may prove useful for the analysis of the genetic control of flower development and morphogenesis in the higher plant.
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Article de synthese decrivant les mutants d'Arabidopsis avec une morphologie modifiee a l'un des 3 niveaux suivants: l'embryogenese, la formation des organes (developpement floral) et le developpement cellulaire (trichomes)
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The chapter reviews the vectors and methods based on the Agrobacterium tumefaciens Ti plasmid. The chapter also describes the construction and use of several new cassette plasmids for the expression of gene-coding sequences in plants. Improvements in the basic vectors and selectable markers for the identification of transformants are appearing at an increasing pace. These are leading to increased efficiency and ease of producing transgenic plants as well as for an extension of the range of plant species, which may be transformed. The Agrobacterium tumefaciens Ti plasmid-derived vectors are the easiest and most utilized of the various schemes for the introduction of DNAs into plants. In nature, A. tumefaciens infects most dicotyledonous and some monocotyledonous plants by entry through wound sites. Methotrexate (mtx) is an antimetabolite that inhibits eukaryotic dihydrofolate reductase (dhfr), ultimately preventing the biosynthesis of glycine, thymine, and purines. The expression cassette vectors described in the chapter are used to express coding sequences for a wide range of bacterial, mammalian, and plant genes.
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Unusual gene interactions were observed in several doubly transformed tobacco plants which were obtained following sequential transformation steps using two T-DNAs encoding different selection and screening markers. The expression of T-DNA-I, which encoded kanamycin resistance (Kanr) and nopaline synthase (NOS), was suppressed in some, but not all, of the double transformants after the introduction of T-DNA-II, which encoded hygromycin resistance (Hygr) and octopine synthase (OCS). Double transformants in which T-DNA-I had been inactivated could produce KanrNOS+ progeny, but these were shown to lack T-DNA-II, thus establishing the role of this T-DNA in the suppression of T-DNA-I. Reversible cytosine methylation of the promoters of T-DNA-I genes was shown to correlate with their activation/inactivation cycle. In this paper we pursue further the questions of the mechanism of suppression of T-DNA-I genes by T-DNA-II, and also the timing and extent of demethylation of T-DNA-I promoters in Kanr progeny following the loss of T-DNA-II. We propose that the suppression is due to the competition between homologous regions on each T-DNA for binding to nuclear sites with fixed locations. We further suggest that incomplete demethylation patterns of T-DNA-I promoters in Kanr progeny reflect the existence in the shoot apex meristem of two cell populations, which have either methylated or unmethylated T-DNA-I promoters, respectively. Thus, Kanr progeny are epigenetic chimeras with respect to the expression of T-DNA-I genes.
Article
We describe a locus, SUPERMAN, mutations in which result in extra stamens developing at the expense of the central carpels in the Arabidopsis thaliana flower. The development of superman flowers, from initial primordium to mature flower, is described by scanning electron microscopy. The development of doubly and triply mutant strains, constructed with superman alleles and previously identified homeotic mutations that cause alterations in floral organ identity, is also described. Essentially additive phenotypes are observed in superman agamous and superman apetala2 double mutants. The epistatic relationships observed between either apetala3 or pistillata and superman alleles suggest that the SUPERMAN gene product could be a regulator of these floral homeotic genes. To test this, the expression patterns of AGAMOUS and APETALA3 were examined in superman flowers. In wild-type flowers, APETALA3 expression is restricted to the second and third whorls where it is required for the specification of petals and stamens. In contrast, in superman flowers, APETALA3 expression expands to include most of the cells that would normally constitute the fourth whorl. This ectopic APETALA3 expression is proposed to be one of the causes of the development of the extra stamens in superman flowers. The spatial pattern of AGAMOUS expression remains unaltered in superman flowers as compared to wild-type flowers. Taken together these data indicate that one of the functions of the wild-type SUPERMAN gene product is to negatively regulate APETALA3 in the fourth whorl of the flower. In addition, superman mutants exhibit a loss of determinacy of the floral meristem, an effect that appears to be mediated by the APETALA3 and PISTILLATA gene products.
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This paper describes a method of transferring fragments of DNA from agarose gels to cellulose nitrate filters. The fragments can then be hybridized to radioactive RNA and hybrids detected by radioautography or fluorography. The method is illustrated by analyses of restriction fragments complementary to ribosomal RNAs from Escherichia coli and Xenopus laevis, and from several mammals.
Article
Mutations in the APETALA3 (AP3) gene of A. thaliana result in homeotic transformations of petals to sepals and stamens to carpels. We have cloned the AP3 gene from Arabidopsis based on its homology to the homeotic flower gene deficiens (DEFA) from the distantly related plant Antirrhinum majus. The sequence of four ap3 mutant alleles and genetic mapping analysis prove that the DEFA homolog is AP3. Like several other plant homeotic genes, the AP3 gene contains a MADS box and likely acts as a transcription factor. The region-specific spatial expression pattern of AP3 rules out certain types of sequential models of flower development and argues in favor of a spatial model based on positional information. Since DEFA and AP3 have very similar protein products, mutant phenotypes, and spatial expression patterns, it is likely that these genes are cognate homologs.
Article
Genetic studies suggest that three homeotic functions, designated A, B, and C, act alone and together to specify the fate of floral organ primordia in distantly related dicotyledonous plant species. To test the genetic model, we have generated transgenic tobacco plants that ectopically express the AGAMOUS gene from Brassica napus, which is necessary for the C function. Flowers on the resulting plants showed homeotic transformations of sepals into carpels and petals into stamens. These phenotypes are consistent with predictions from the genetic model, show that expression of AGAMOUS is sufficient to provide ectopic C function, and demonstrate that the structure of flowers can be manipulated in a predictable manner by altering the expression of a single regulatory gene. Furthermore, the generation of the predicted transformations by ectopic expression of the Brassica gene in transgenic tobacco indicates that gene functions are interchangeable between phylogenetically distant species.
Article
Anomalous flowering of the Antirrhinum majus mutant squamosa (squa) is characterized by excessive formation of bracts and the production of relatively few and often malformed or incomplete flowers. To study the function of squamosa in the commitment of an inflorescence lateral meristem to floral development, the gene was cloned and its genomic structure, a well as that of four mutant alleles, was determined. SQUA is a member of a family of transcription factors which contain the MADS-box, a conserved DNA binding domain. In addition, we analysed the temporal and spatial expression pattern of the squa gene. Low transcriptional activity of squa is detectable in bracts and in the leaves immediately below the inflorescence. High squa transcript levels are seen in the inflorescence lateral meristems as soon as they are formed in the axils of bracts. Squa transcriptional activity persists through later stages of floral morphogenesis, with the exception of stamen differentiation. Although necessary for shaping a normal racemose inflorescence, the squa function is not absolutely essential for flower development. We discuss the function of the gene during flowering, its likely functional redundancy and its possible interaction with other genes participating in the genetic control of flower formation in Antirrhinum.
Article
We characterized the distribution of AGAMOUS (AG) RNA during early flower development in Arabidopsis. Mutations in this homeotic gene cause the transformation of stamens to petals in floral whorl 3 and of carpels to another ag flower in floral whorl 4. We found that AG RNA is present in the stamen and carpel primordia but is undetectable in sepal and petal primordia throughout early wild-type flower development, consistent with the mutant phenotype. We also analyzed the distribution of AG RNA in apetela2 (ap2) mutant flowers. AP2 is a floral homeotic gene that is necessary for the normal development of sepals and petals in floral whorls 1 and 2. In ap2 mutant flowers, AG RNA is present in the organ primordia of all floral whorls. These observations show that the expression patterns of the Arabidopsis floral homeotic genes are in part established by regulatory interactions between these genes.
Article
Five genes with homology to the floral homeotic genes deficiens of Antirrhinum and agamous of Arabidopsis were isolated from tomato. Each of the five genes is unique in the genome and could be localized to a different chromosome by RFLP mapping. Four of the tomato genes (hereafter TM) are flower-specific with distinguishable temporal expression. TM4 and TM8 are 'early', while TM5 and TM6 are 'late' genes. TM4 is homologous to squamous and TM6 is similar to deficiens, which are, respectively, 'early' and 'late' bona fide homeotic genes in Antirrhinum. The proteins encoded by the five tomato genes, like several known homeotic genes from other plants, contain within their N-terminus a highly conserved DNA-binding domain, the MADS box. All known plant MADS box genes also share, however, other properties. They all contain a central, moderately conserved, and rather basic domain, and a highly divergent or even missing C-terminal domain. Furthermore, molecular modelling predicts the presence of a conserved amphipatic alpha helix, at a constant distance from the MADS box in each of these proteins. The common properties of eight MADS box proteins from three plant families indicate that all their domains were coded for by the same ancestor gene. The sequence homology between pairs of MADS genes from different species indicates that the MADS ancestor gene multiplied and diverged in an ancestor plant common to several dicotyledon families.
Article
Plants carrying the floricaula (flo) mutation cannot make the transition from inflorescence to floral meristems and have indeterminate shoots in place of flowers. The flo-613 allele carries a Tam3 transposon insertion, which allowed the isolation of the flo locus. The flo gene encodes a putative protein (FLO) containing a proline-rich N-terminus and a highly acidic region. In situ hybridization shows that the flo gene is transiently expressed in the very early stages of flower development. The earliest expression seen is in bract primordia, followed by sepal, petal, and carpel primordia, but no expression is detected in stamen primordia. This pattern of expression has implications for how flo affects phyllotaxis, organ identity, and determinacy. We propose that flo interacts in a sequential manner with other homeotic genes affecting floral organ identity.
Article
The specialized sets of genes that determine different cell types in yeast are controlled by combinations of DNA-binding proteins some of which are present only in certain cell types whereas others are present in all cell types. Final differentiation requires an inductive signal that triggers both gene transcription and cell-cycle arrest. Synthesis of the proteins coded by the 'master regulatory' mating-type locus is regulated so as to generate a heterogeneous mitotic cell population containing a stem-cell lineage.
Article
We previously reported the isolation of yeast mutants that seem to affect the function of certain autonomously replicating sequences (ARSs). These mutants are known as mcm for their defect in the maintenance of minichromosomes. We have now characterized in more detail one ARS-specific mutation, mcm1-1. This Mcm1 mutant has a second phenotype; MAT alpha mcm1-1 strains are sterile. MCM1 is non-allelic to other known alpha-specific sterile mutations and, unlike most genes required for mating, it is essential for growth. The alpha-specific sterile phenotype of the mcm1-1 mutant is manifested by its failure to produce a normal amount of the mating pheromone, alpha-factor. In addition, transcripts of the MF alpha 1 and STE3 genes, which encode the alpha-factor precursor and the alpha-factor receptor, respectively, are greatly reduced in this mutant. These and other properties of the mcm1-1 mutant suggest that the MCM1 protein may act as a transcriptional activator of alpha-specific genes. We have cloned, mapped and sequenced the wild-type and mutant alleles of MCM1, which is located on the right arm of chromosome XIII near LYS7. The MCM1 gene product is a protein of 286 amino acid residues and contains an unusual region in which 19 out of 20 residues are either aspartic or glutamic acid, followed by a series of glutamine tracts. MCM1 has striking homology to ARG80, a regulatory gene of the arginine metabolic pathway located about 700 base-pairs upstream from MCM1. A substitution of leucine for proline at amino acid position 97, immediately preceding the polyanionic region, was shown to be responsible for both the alpha-specific sterile and minichromosome-maintenance defective phenotypes of the mcm1-1 mutant.
Article
The serum response element (SRE) is a sequence required for transient transcriptional activation of genes in response to growth factors. We have isolated cDNA clones encoding serum response factor (SRF), a ubiquitous nuclear protein that binds to the SRE. The SRF gene is highly conserved through evolution, and in cultured cells its transcription is itself transiently increased following serum stimulation. A cDNA clone of SRF expressed in vitro generates protein that forms complexes indistinguishable from those formed with HeLa cell SRF, as judged by DNA binding specificity and the ability to promote SRE-dependent in vitro transcription. SRF binds DNA as a dimer, and the DNA binding/dimerization domain of the protein exhibits striking homology to two yeast regulatory proteins.
Article
Although promoter regions for many plant nuclear genes have been sequenced, identification of the active promoter sequence has been carried out only for the octopine synthase promoter. That analysis was of callus tissue and made use of an enzyme assay. We have analysed the effects of 5' deletions in a plant viral promoter in tobacco callus as well as in regenerated plants, including different plant tissues. We assayed the RNA transcription product which allows a more direct assessment of deletion effects. The cauliflower mosaic virus (CaMV) 35S promoter provides a model plant nuclear promoter system, as its double-strand DNA genome is transcribed by host nuclear RNA polymerase II from a CaMV minichromosome. Sequences extending to -46 were sufficient for accurate transcription initiation whereas the region between -46 and -105 increased greatly the level of transcription. The 35S promoter showed no tissue-specificity of expression.
Article
We present the initial phenotypic characterization of an Arabidopsis mutation, terminal flower 1-1 (tfl1-1), that identifies a new genetic locus, TFL1. The tfl1-1 mutation causes early flowering and limits the development of the normally indeterminate inflorescence by promoting the formation of a terminal floral meristem. Inflorescence development in mutant plants often terminates with a compound floral structure consisting of the terminal flower and one or two subtending lateral flowers. The distal-most flowers frequently contain chimeric floral organs. Light microscopic examination shows no structural aberrations in the vegetative meristem or in the inflorescence meristem before the formation of floral buttresses. The wild-type appearance of lateral flowers and observations of double mutant combinations of tfl1-1 with the floral morphogenesis mutations apetala 1-1 (ap1-1), ap2-1, and agamous (ag) suggest that the tfl1-1 mutation does not affect normal floral meristems. Secondary flower formation usually associated with the ap1-1 mutation is suppressed in the terminal flower, but not in the lateral flowers, of tfl1-1 ap1-1 double mutants. Our results suggest that tfl1-1 perturbs the establishment and maintenance of the inflorescence meristem. The mutation lies on the top arm of chromosome 5 approximately 2.8 centimorgans from the restriction fragment length polymorphism marker 217.
Article
We attempted to overexpress chalcone synthase (CHS) in pigmented petunia petals by introducing a chimeric petunia CHS gene. Unexpectedly, the introduced gene created a block in anthocyanin biosynthesis. Forty-two percent of plants with the introduced CHS gene produced totally white flowers and/or patterned flowers with white or pale nonclonal sectors on a wild-type pigmented background; none of hundreds of transgenic control plants exhibited such phenotypes. Progeny testing of one plant demonstrated that the novel color phenotype co-segregated with the introduced CHS gene; progeny without this gene were phenotypically wild type. The somatic and germinal stability of the novel color patterns was variable. RNase protection analysis of petal RNAs isolated from white flowers showed that, although the developmental timing of mRNA expression of the endogenous CHS gene was not altered, the level of the mRNA produced by this gene was reduced 50-fold from wild-type levels. Somatic reversion of plants with white flowers to phenotypically parental violet flowers was associated with a coordinate rise in the steady-state levels of the mRNAs produced by both the endogenous and the introduced CHS genes. Thus, in the altered white flowers, the expression of both genes was coordinately suppressed, indicating that expression of the introduced CHS gene was not alone sufficient for suppression of endogenous CHS transcript levels. The mechanism responsible for the reversible co-suppression of homologous genes in trans is unclear, but the erratic and reversible nature of this phenomenon suggests the possible involvement of methylation.
Article
Culture conditions were developed that induce Arabidopsis thaliana (L.) Heynh. root cuttings to regenerate shoots rapidly and at 100% efficiency. The shoots produce viable seeds in vitro or after rooting in soil. A transformation procedure for Arabidopsis root explants based on kanamycin selection was established. By using this regeneration procedure and an Agrobacterium tumor-inducing Ti plasmid carrying a chimeric neomycin phosphotransferase II gene (neo), transformed seed-producing plants were obtained with an efficiency between 20% and 80% within 3 months after gene transfer. F(1) seedlings of these transformants showed Mendelian segregation of the kanamycin-resistance trait. The transformation method could be applied to three different Arabidopsis ecotypes. In addition to the neo gene, a chimeric bar gene conferring resistance to the herbicide Basta was introduced into Arabidopsis. The expression of the bar gene was shown by enzymatic assay.
Article
Homeotic mutants have been useful for the study of animal development. Such mutants are also known in plants. The isolation and molecular analysis of several homeotic genes in Antirrhinum majus provide insights into the underlying molecular regulatory mechanisms of flower development. A model is presented of how the characteristic sequential pattern of developing organs, comprising the flower, is established in the process of morphogenesis.
Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter
  • Odell
The MADS box gene family in tomato: temporal expression during floral development, conserved secondary structures and homology with homeotic genes from Antirrhinum and Arabidopsis
  • Pnueli