Flower Meristem Research Articles

Evaluation of seven promoters to achieve germline directed Cre-lox recombination in Arabidopsis thaliana

Site-specific recombination systems, such as Cre-lox from bacteriophage P1, have become very important tools for plant genome engineering. In many cases a constitutive promoter is used to express the recombinase gene. However, for certain research and commercial applications constitutive Cre-mediated recombination may not be desirable. We have evaluated the potential of seven different germline promoter:cre fusions to remove a stably integrated lox cassette through Cre-mediated recombination in Arabidopsis thaliana. We monitored the functionality of each promoter in the germline of primary transformants by analyzing the presence of the recombined lox cassette in T(2) progeny. The selected germline promoters are involved in different developmental cues, including early stem cell identity (CLAVATA3), flower meristem identity (LEAFY, APETALA1), floral organ identity (AGAMOUS), and meiosis (SOLO DANCERS, DMC1, SWITCH1). For five out of these seven promoters we were able to show that efficient Cre-mediated recombination does, indeed, occur and that the recombination takes place at some point during germline development. Furthermore, a recombination efficiency of 100% is obtained when Cre-expression is regulated by the CLAVATA3 promoter. In addition, with these promoters, we observe much less variation in recombination frequency than previously reported for the 35S promoter. For these reasons, we believe that germline-specific Cre-lox recombination provides an additional tool to the site-specific recombination technology in plants.

Temporal and Spatial Requirement of EMF1 Activity for Arabidopsis Vegetative and Reproductive Development

EMBRYONIC FLOWER (EMF) genes are required to maintain vegetative development via repression of flower homeotic genes in Arabidopsis. Removal of EMF gene function caused plants to flower upon germination, producing abnormal and sterile flowers. The pleiotropic effect of emf1 mutation suggests its requirement for gene programs involved in diverse developmental processes. Transgenic plants harboring EMF1 promoter::glucuronidase (GUS) reporter gene were generated to investigate the temporal and spatial expression pattern of EMF1. These plants displayed differential GUS activity in vegetative and flower tissues, consistent with the role of EMF1 in regulating multiple gene programs. EMF1::GUS expression pattern in emf mutants suggests organ-specific auto-regulation. Sense- and antisense (as) EMF1 cDNA were expressed under the control of stage- and tissue-specific promoters in transgenic plants. Characterization of these transgenic plants showed that EMF1 activity is required in meristematic as well as differentiating tissues to rescue emf mutant phenotype. Temporal removal or reduction of EMF1 activity in the embryo or shoot apex of wild-type seedlings was sufficient to cause early flowering and terminal flower formation in adult plants. Such reproductive cell memory is reflected in the flower MADS-box gene activity expressed prior to flowering in these early flowering plants. However, temporal removal of EMF1 activity in flower meristem did not affect flower development. Our results are consistent with EMF1's primary role in repressing flowering in order to allow for vegetative growth.

A role for Arabidopsis PUCHI in floral meristem identity and bract suppression.

At the onset of flowering, the Arabidopsis thaliana primary inflorescence meristem starts to produce flower meristems on its flank. Determination of floral fate is associated with changes in the growth pattern and expression of meristem identity genes and suppression of a subtending leaf called a bract. Here, we show a role in floral fate determination and bract suppression for the PUCHI gene, an AP2/EREBP family gene that has previously been reported to play roles in lateral root morphogenesis. Mutations in PUCHI cause partial conversion of flowers to inflorescences, indicating that PUCHI is required for flower meristem identity. PUCHI is transiently expressed in the early flower meristem and accelerates meristem bulging while it prevents the growth of the bract primordium. The function of PUCHI in floral fate determination and bract suppression overlaps that of the BLADE-ON-PETIOLE1 (BOP1) and BOP2 genes, which encode a pair of redundant regulatory proteins involved in various developmental processes, including leaf morphogenesis and flower patterning. We also show that PUCHI acts together with BOP1 and BOP2 to promote expression of LEAFY and APETALA1, two central regulators of floral meristem identity. Expression patterns of the PUCHI and BOP genes point to a role in spatial control of flower-specific activation of these meristem identity genes.

BAM receptors regulate stem cell specification and organ development through complex interactions with CLAVATA signaling.

The CLAVATA1 (CLV1) receptor kinase regulates stem cell specification at shoot and flower meristems of Arabidopsis. Most clv1 alleles are dominant negative, and clv1 null alleles are weak in phenotype, suggesting additional receptors functioning in parallel. We have identified two such parallel receptors, BAM1 and BAM2. We show that the weak nature of the phenotype of clv1 null alleles is dependent on BAM activity, with bam clv mutants exhibiting severe defects in stem cell specification. Furthermore, BAM activity in the meristem depends on CLV2, which is required in part for CLV1 function. In addition, clv1 mutants enhance many of the Bam(-) organ phenotypes, indicating that, contrary to current understanding, CLV1 function is not specific to the meristem. CLV3 encodes a small, secreted peptide that acts as the ligand for CLV1. Mutations in clv3 lead to increased stem cell accumulation. Surprisingly, bam1 and bam2 mutants suppress the phenotype of clv3 mutants. We speculate that in addition to redundant function in the meristem center, BAM1 and BAM2 act to sequester CLV3-like ligands in the meristem flanks.

Structural basis for LEAFY floral switch function and similarity with helix-turn-helix proteins

The LEAFY (LFY) protein is a key regulator of flower development in angiosperms. Its gradually increased expression governs the sharp floral transition, and LFY subsequently controls the patterning of flower meristems by inducing the expression of floral homeotic genes. Despite a wealth of genetic data, how LFY functions at the molecular level is poorly understood. Here, we report crystal structures for the DNA-binding domain of Arabidopsis thaliana LFY bound to two target promoter elements. LFY adopts a novel seven-helix fold that binds DNA as a cooperative dimer, forming base-specific contacts in both the major and minor grooves. Cooperativity is mediated by two basic residues and plausibly accounts for LFY's effectiveness in triggering sharp developmental transitions. Our structure reveals an unexpected similarity between LFY and helix-turn-helix proteins, including homeodomain proteins known to regulate morphogenesis in higher eukaryotes. The appearance of flowering plants has been linked to the molecular evolution of LFY. Our study provides a unique framework to elucidate the molecular mechanisms underlying floral development and the evolutionary history of flowering plants.

Expression analysis of ANTHOCYANINLESS2 gene in Arabidopsis

The ANTHOCYANINLESS2 ( ANL2) gene of Arabidopsis thaliana affects the accumulation of anthocyanin in subepidermal cells and the organization of cells in the primary root, suggesting that this homeobox gene determines the nature of epidermis and/or subepidermis. In situ hybridization showed that ANL2 mRNA accumulated in peripheral region of young leaves, flower meristems, anthers and pistils. Promoter activity of ANL2 was also examined by using uidA as a reporter. β-Glucuronidase (GUS) was expressed in the shoot apex, young leaves, root tips, anthers and ovules. In rosette leaves, subepidermal tissues showed strong GUS expression, while epidermal cells including guard cells and trichome had weak GUS expression. In the primary root, GUS was expressed in epidermal and lateral root cap cells. In the rosette leaf, the pattern of GUS expression was correlated with the pattern of anthocyanin accumulation in the subepidermis but not in epidermis. The expression of ANL2 was not affected by irradiation and the defects in TTG1.

An exploration of LEAFY expression in independent evolutionary origins of rosette flowering in Brassicaceae

Whereas most Brassicaceae produce flowers on an elongated inflorescence, a few lineages produce flowers directly from the vegetative rosette on elongated pedicels. Knowing the extent to which independent origins of rosette flowering involve the same developmental and genetic mechanisms could clarify the constraints acting on plant architectural evolution. Prior work in Idahoa, Ionopsidium, and Leavenworthia suggested that changes in the activity or expression of the flower meristem identity gene, LEAFY (LFY), played a role in all three origins of rosette flowering. Here we studied the developmental morphology of L. crassa and immunolocalization of LFY protein in Leavenworthia and Ionopsidium to further compare independent origins of rosette flowering. Leavenworthia crassa differs from Ionopsidium and Idahoa in producing ebracteate flowers. Flowers are, however, associated with "squamules," here interpreted as stipules of a cryptic bract. LFY was detected in L. crassa flower primordia but not in inflorescence meristems. In contrast, the rosette flowering Io. acaule accumulated LFY protein in the inflorescence meristem, whereas its inflorescence-flowering close relative, Io. prolongoi, did not. Thus, although different cases of rosette flowering likely entailed modifications of the same meristem identity program, distinct developmental genetic mechanisms appear to be involved in each case.

Regulatory networks that function to specify flower meristems require the function of homeobox genes PENNYWISE and POUND‐FOOLISH in Arabidopsis

Flowering is a major developmental phase change that transforms the fate of the shoot apical meristem (SAM) from a leaf-bearing vegetative meristem to that of a flower-producing inflorescence meristem. In Arabidopsis, floral meristems are specified on the periphery of the inflorescence meristem by the combined activities of the FLOWERING LOCUS T (FT)-FD complex and the flower meristem identity gene, LEAFY (LFY). Two redundant functioning homeobox genes, PENNYWISE (PNY) and POUND-FOOLISH (PNF), which are expressed in the vegetative and inflorescence SAM, regulate patterning events during reproductive development, including floral specification. To determine the role of PNY and PNF in the floral specification network, we characterized the genetic relationship of these homeobox genes with LFY and FT. Results from this study demonstrate that LFY functions downstream of PNY and PNF. Ectopic expression of LFY promotes flower formation in pny pnf plants, while the flower specification activity of ectopic FT is severely attenuated. Genetic analysis shows that when mutations in pny and pnf genes are combined with lfy, a synergistic phenotype is displayed that significantly reduces floral specification and alters inflorescence patterning events. In conclusion, results from this study support a model in which PNY and PNF promote LFY expression during reproductive development. At the same time, the flower formation activity of FT is dependent upon the function of PNY and PNF.

Expression Analysis of the LFY and TFL1 Homologs in Floral Buds of Japanese Pear (Pyrus pyrifolia Nakai) and Quince (Cydonia oblonga Mill.)

We investigated the expression of Japanese pear (Pyrus pyrifolia) and quince (Cydonia oblonga) homologs of Arabidopsis thaliana flowering-related genes LEAFY (LFY) and TERMINAL FLOWER 1 (TFL1) in dissected floral tissues. Japanese pear and quince belong to the subfamily Maloideae (Rosaceae), which is an ancient polyploid; thus, the genome of each plant contains at least two different homologs of LFY and TFL1. We examined the expression of each homolog separately using gene-specific reverse transcription-polymerase chain reaction (RT-PCR). Under the experimental conditions used in this study, there was no difference in the expression pattern of LFY homologs, regardless of the species and homolog type. In contrast, TFL1 homologs were differentially expressed in Japanese pear and quince. In Japanese pear, PpTFL1-1 and PpTFL1-2 were expressed in the apical meristem prior to floral differentiation; however, during floral differentiation, PpTFL1-2 expression disappeared and PpTFL1-1 alone continued to be expressed in the developing inflorescence. In quince, CoTFL1-1 was the only TFL1 homolog detected prior to floral differentiation. Upon floral differentiation, both CoTFL1-1 and CoTFL1-2 were expressed mainly in the domed apical meristem. These expressions decreased to an undetectable level once the apical meristem had transformed into a single flower meristem. Thus, Japanese pear and quince TFL1 homologs are supposed to be involved in the formation of domed apical meristems during early reproductive development. Our results imply the importance of PpTFL1-1 in morphological development of Japanese pear inflorescence.

Ectopic expression of a hyacinth AGL6 homolog caused earlier flowering and homeotic conversion in Arabidopsis

MADS-box genes are involved in floral organ development. Here we report that an AGL6(Agamous-like 6)-like MADS-box gene, HoAGL6, was isolated from Hyacinthus orientalis L. Expression pattern analysis demonstrated that HoAGL6 transcript was detected in inflorescence buds, tepals, carpels and ovules, but not in stamina, leaves or scales. Transgenic Arabidopsis plants ectopically expressing HoAGL6 exhibited novel phenotypes of significantly reduced plant size, extremely early flowering, and losing inflorescence indeterminacy. In addition, wide homeotic conversion of sepals, petals, and leaves into carpel-like or ovary structures, and disappearance or number reduction of stamens in 35S::HoAGL6 Arabidopsis plants were also observed. RT-PCR analysis indicated that the expressions of flowering time gene SOC1 and flower meristem identity gene LFY were significantly up-regulated in 35S::HoAGL6 transgenic Arabidopsis plants, and the expression levels of floral organ identity genes AG and SEP1 in leaves were also elevated. These results indicated that HoAGL6 was involved in the regulation of flower transition and flower organ formation.

Effect of plant growth regulators on floral differentiation and seed production in Jojoba ( Simmondsia chinensis (Link) Schneider)

The effect of foliar application of cytokinin (benzyl-adenine, 150 mg L −1) and gibberellin (GA 4 + GA 7, 150 mg L −1) on growth and flower development of 5-year-old plants of two jojoba clones was studied. The plant growth regulators were applied on October 5, 1999 (spring) and the plants were evaluated 120, 240 and 360 days after application. Shoot length, total number of nodes and number of nodes with branching were statistically different between clones but not between the growth regulator treatments. The total number of flowers on both clones was significantly increased by treatment with benzyl-adenine (BA) and significantly reduced by treatment with gibberellin. The seed yields, evaluated 180 days after application, were not statistically different from the control due to an increase in flower abortion. One clone treated with gibberellin showed a significant decrease in number and weight of seeds, the other did not. Histology of axillary buds revealed that BA application on one clone (4.11.32) enlarged the flower meristem, differentiating multiple flower production.

Flowering transition in grapevine (Vitis viniferaL.)This review is one of a selection of papers presented at the symposium onVitisat the XVII International Botanical Congress held in Vienna, Austria, 2005.

The available information on the regulation of flowering transition in model systems, such as Arabidopsis and rice, provides a framework to undertake the study of this process in plant species with different growth strategies. The grapevine ( Vitis vinifera L.) is the most widely cultivated and economically important fruit crop in the world. Understanding the regulation of flowering transition in this species can be relevant for the improvement of yield and quality of the crop. The grapevine is a representative of the family Vitaceae, whose species mostly grow as vines and have evolved climbing organs, tendrils, which are ontogenetically related to the reproductive organs. Here, we summarize the available information on the flowering transition in the grapevine. With this purpose, we first describe the vegetative and reproductive development of the grapevine as well as the reports on the physiology of flowering induction in this species. As well, we review the recent information on the molecular genetics of flowering signal integrator and flower meristem identity genes in the grapevine and compare the process with what is already known in model systems such as Arabidopsis. Finally, we propose a preliminary model to explain the regulation of flower initiation in the grapevine that is useful to identify its differential features and infer future prospects in the understanding of this process.

Diversification and co‐option of RAD‐like genes in the evolution of floral asymmetry

To understand how changes in gene regulatory networks lead to novel morphologies, we have analysed the evolution of a key target gene, RAD, controlling floral asymmetry. In Antirrhinum, flower asymmetry depends on activation of RAD in dorsal regions of the floral meristem by the upstream regulators CYC and DICH. We show that Arabidopsis, a species with radially symmetric flowers, contains six RAD-like genes, reflecting at least three duplications since the divergence of Antirrhinum and Arabidopsis. Unlike the situation in Antirrhinum, none of the Arabidopsis RAD-like genes are activated in dorsal regions of the flower meristem. Rather, the RAD-like genes are expressed in distinctive domains along radial or ab-adaxial axes, consistent with a range of developmental roles. Introduction of a RAD genomic clone from Antirrhinum into Arabidopsis leads to a novel expression pattern that is distinct from the expression pattern of RAD in Antirrhinum and from the endogenous RAD-like genes of Arabidopsis. Nevertheless, RAD is able to influence developmental targets in Arabidopsis, as ectopic expression of RAD has developmental effects in this species. Taken together, our results suggest that duplication and divergence of RAD-like genes has involved a range of cis- and trans-regulatory changes. It is possible that such changes led to the coupling of RAD to CYC regulation in the Antirrhinum lineage and hence the co-option of RAD had a role in the generation of flower dorsoventral asymmetry.

Comparison of Early Inflorescence Development between Japanese Pear (Pyrus pyrifolia Nakai) and Quince (Cydonia oblonga Mill.)

Japanese pear (Pyrus pyrifolia) and quince (Cydonia oblonga) form different inflorescence architectures, the former forms raceme inflorescence with about eight flowers and the latter forms solitary-flowered inflorescence. We observed the floral differentiation of Japanese pear and quince to clarify the differences in their early inflorescence development. Floral differentiation of Japanese pear occurred in late June. After apical meristem turned to a dome-like structure, inflorescence developed by forming lateral flower meristems in axils of bracts. The apex of inflorescence became the terminal flower meristem, two or three meristems were initiated in axils of outer bracts and leaf primordia, and finally approximately eight flower meristems were formed in the inflorescence. Floral differentiation of quince was initiated from late October to November after eight leaf primordia had been initiated. Apical meristem transformed to a dome-like structure and initiated sepal primordia. Axillary meristems of bracts or leaf primordia in Japanese pear differentiate to flower meristem while those in quince remained undifferentiated to form axillary buds in the following growing season, resulting in an inflorescence architecture difference in Japanese pear and quince.

Relationship between Floral Development and Transcription Levels of LEAFY and TERMINAL FLOWER 1 Homologs in Japanese Pear (Pyrus pyrifolia Nakai) and Quince (Cydonia oblonga Mill.)

Using real-time reverse-transcription polymerase chain reaction (real-time RT-PCR) and in situ hybridization, we investigated the temporal and spatial expression of LEAFY (LFY) and TERMINAL FLOWER 1 (TFL1) homologs in Japanese pear (Pyrus pyrifolia) and quince (Cydonia oblonga) buds, in order to elucidate their roles in the flower and inflorescence development of fruit trees in the subfamily Maloideae (Rosaceae). Japanese pear and quince were selected because they are very close phylogenetically but develop distinct inflorescence architectures. Floral differentiation in Japanese pear began in late June to early July in Osaka, Japan, forming a raceme inflorescence with about eight flowers, whereas that in quince took place from late October to November in Nagano, Japan, forming a solitary flower in each floral bud. LFY homolog expression levels in both species increased at the floral differentiation stage and remained at relatively high levels in flower meristems after the flower organ differentiation stage. In contrast, TFL1 homolog expression levels in both species were high in vegetative-stage buds, but decreased significantly just before floral differentiation. Japanese pear TFL1 homologs were expressed in the subepidermal layer of the apical meristem before floral differentiation, whereas those of quince were expressed in the epidermal layer of the apical meristem and leaf primordia. We discuss the possible involvement of maloid LFY and TFL1 homologs in triggering floral differentiation, as well as the involvement of the different spatial expression patterns of TFL1 homologs in the inflorescence architectures of Japanese pear and quince.

A genetic framework for flowering-time pathways in Citrus spp.

Floral transition is one the most drastic changes occurring during the life cycle of a plant. The shoot apical meristem switches from the production of leaves with associated secondary shoot meristems to the production of flower meristems. This transition is abrupt and generally irreversible, suggesting it is regulated by a robust gene regulatory network capable of driving sharp transitions. The moment at which this transition occurs is precisely determined by environmental and endogenous signals. A large number of genes acting within these pathways have been cloned in model herbaceous plants such as Arabidopsis thaliana. In this paper, we report the results of our search in the Citrus expressed sequence tag (CitEST) database for expressed sequence tags (ESTs) showing sequence homology with known elements of flowering-time pathways. We have searched all sequence clusters in the CitEST database and identified more than one hundred Citrus spp sequences that codify putative conserved elements of the autonomous, vernalization, photoperiod response and gibberelic acid-controlled flowering-time pathways. Additionally, we have characterized in silico putative members of the Citrus spp homologs to the Arabidopsis CONSTANS family of transcription factors.

The FT/TFL1 gene family in grapevine

The FT/TFL1 gene family encodes proteins with similarity to phosphatidylethanolamine binding proteins which function as flowering promoters and repressors. We show here that the FT/TFL1 gene family in Vitis vinifera is composed of at least five genes. Sequence comparisons with homologous genes identified in other dicot species group them in three major clades, the FT, MFT and TFL1 subfamilies, the latter including three of the Vitis sequences. Gene expression patterns are in agreement with a role of VvFT and VvMFT as flowering promoters; while VvTFL1A, VvTFL1B and VvTFL1C could be associated with vegetative development and maintenance of meristem indetermination. Overexpression of VvFT in transgenic Arabidopsis plants generates early flowering phenotypes similar to those produced by FT supporting a role for this gene in flowering promotion. Overexpression of VvTFL1A does not affect flowering time but the determination of flower meristems, strongly altering inflorescence structure, which is consistent with the biological roles assigned to similar genes in other species.

Reproductive meristem fates in Gerbera

Flowering plants go through several phases between regular stem growth and the actual production of flower parts. The stepwise conversion of vegetative into inflorescence and floral meristems is usually unidirectional, but under certain environmental or genetic conditions, meristems can revert to an earlier developmental identity. Vegetative meristems are typically indeterminate, producing organs continuously, whereas flower meristems are determinate, shutting down their growth after reproductive organs are initiated. Inflorescence meristems can show either pattern. Flower and inflorescence development have been investigated in Gerbera hybrida, an ornamental plant in the sunflower family, Asteraceae. Unlike the common model species used to study flower development, Gerbera inflorescences bear a fixed number of flowers, and the architecture of the flowers differ in that Gerbera ovaries are inferior (borne below the perianth). This architectural difference has been exploited to show that floral meristem determinacy and identity are spatially and genetically distinct in Gerbera, and we have shown that a single SEPALLATA-like MADS domain factor controls both flower and inflorescence meristem fate in the plant. Although these phenomena have not been directly observed in Arabidopsis, the integrative role of the SEPALLATA function in reproductive meristem development may be general for all flowering plants.

GA4 Is the Active Gibberellin in the Regulation ofLEAFYTranscription andArabidopsisFloral Initiation

Flower initiation in Arabidopsis thaliana under noninductive short-day conditions is dependent on the biosynthesis of the plant hormone gibberellin (GA). This dependency can be explained, at least partly, by GA regulation of the flower meristem identity gene LEAFY (LFY) and the flowering time gene SUPPRESSOR OF CONSTANS1. Although it is well established that GA(4) is the active GA in the regulation of Arabidopsis shoot elongation, the identity of the GA responsible for the regulation of Arabidopsis flowering has not been established. Through a combination of GA quantifications and sensitivity assays, we show that GA(4) is the active GA in the regulation of LFY transcription and Arabidopsis flowering time under short-day conditions. The levels of GA(4) and sucrose increase dramatically in the shoot apex shortly before floral initiation, and the regulation of genes involved in GA metabolism suggests that this increase is possibly due to transport of GAs and sucrose from outside sources to the shoot apex. Our results demonstrate that in the dicot Arabidopsis, in contrast with the monocot Lolium temulentum, GA(4) is the active GA in the regulation of both shoot elongation and flower initiation.

Unique and redundant functional domains of APETALA1 and CAULIFLOWER, two recently duplicated Arabidopsis thaliana floral MADS-box genes

APETALA1 (AP1) and CAULIFLOWER (CAL) are closely related MADS box genes that are partially redundant during Arabidopsis thaliana floral meristem determination. AP1 is able to fully substitute for CAL functions, but not vice versa, and AP1 has unique sepal and petal identity specification functions. In this study, the unique and redundant functions of these two genes has been mapped to the four protein domains that characterize type-II MADS-domain proteins by expressing all 15 chimeric combinations of AP1 and CAL cDNA regions under control of the AP1 promoter in ap1-1 loss-of-function plants. The "in vivo" function of these chimeric genes was analysed in Arabidopsis plants by expressing the chimeras. Rescue of flower meristem and sepal/petal identities was scored in single and multiple insert homozygous transgenic lines. Using these chimeric lines, it was found that distinct residues of the AP1 K domain not shared by the same CAL domain are necessary and sufficient for complete recovery of floral meristem identity, in the context of the CAL protein sequence, while both AP1 COOH and K domains are indispensable for complete rescue of sepal identity. By contrast, either one of these two AP1 domains is necessary and sufficient for complete petal identity recovery. It was also found that there were positive and negative synergies among protein domains and their combinations, and that multiple-insert lines showed relatively better rescue than equivalent single-insert lines. Finally, several lines had flowers with extra sepals and petals suggesting that chimeric proteins yield abnormal transcriptional complexes that may alter the expression or regulation of genes that control floral organ number under normal conditions.