RESUMO
In many plants, timing of flowering is regulated by day length. In Arabidopsis, florigen, FLOWERING LOCUS T (FT) protein, is synthesized in leaf phloem companion cells in response to long days and is transported to the shoot apical meristem (SAM) through the phloem. The temporal aspects of florigen transportation have been studied in various plants by physiological experiments. Nevertheless, little is known about how FT protein transportation is regulated in Arabidopsis. In this study, we performed heat shock-based transient FT induction in a single leaf blade and detected the FT protein in the shoot apex by 2D-PAGE. We demonstrated that detectable amounts of FT were transported from the leaf to the shoot apex within 8 h, and subsequent FT-induced target gene expression was detected within 8-12 h. Furthermore, we identified three amino acid residues (V70, S76 and R83) where missense mutations led to reduced mobility. Interestingly, these FT variants lost only their transportation ability, but retained their flowering promotion capacity, suggesting that discrete amino acids are involved in flowering regulation and transport regulation. Since the interaction with FT-INTERACTING PROTEIN 1 (FTIP1) was not affected in these FT variants, we hypothesize that the three amino acid residues are not involved in the FTIP1-mediated pathway of uploading, but rather in the subsequent step(s) of FT transport.
Assuntos
Florígeno/metabolismo , Flores/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana/metabolismo , Meristema/metabolismo , Mutação , Floema/metabolismo , Transporte Proteico/fisiologiaRESUMO
Plant architecture shows a large degree of developmental plasticity. Some of the key determinants are the timing of the floral transition induced by a systemic flowering signal (florigen) and the branching pattern regulated by key factors such as BRANCHED1 (BRC1). Here, we report that BRC1 interacts with the florigen proteins FLOWERING LOCUS T (FT) and TWIN SISTER OF FT (TSF) but not with TERMINAL FLOWER1, a floral repressor. FT protein induced in leaves moves into the subtended bud, suggesting that FT protein also plays a role in promotion of the floral transition in the axillary meristem (AM). The brc1-2 mutant shows an earlier floral transition in the axillary shoots compared with the wild type, suggesting that BRC1 plays a role in delaying the floral transition of the AMs. Genetic and gene expression analyses suggest that BRC1 interferes with florigen (FT and TSF) function in the AMs. Consistent with this, BRC1 ectopically expressed in the shoot apical meristem delays the floral transition in the main shoot. These results taken together suggest that BRC1 protein interacts with FT and TSF proteins and modulates florigen activity in the axillary buds to prevent premature floral transition of the AMs.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Flores/genética , Meristema/genética , Fatores de Transcrição/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Florígeno/metabolismo , Flores/crescimento & desenvolvimento , Flores/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Mutação , Proteína de Ligação a Fosfatidiletanolamina/genética , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Ligação Proteica , Transporte Proteico , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
Inflorescence structures result from the activities of meristems, which coordinate both the renewal of stem cells in the center and organ formation at the periphery. The fate of a meristem is specified at its initiation and changes as the plant develops. During rice inflorescence development, newly formed meristems acquire a branch meristem (BM) identity, and can generate further meristems or terminate as spikelets. Thus, the form of rice inflorescence is determined by a reiterative pattern of decisions made at the meristems. In the dominant gain-of-function mutant tawawa1-D, the activity of the inflorescence meristem (IM) is extended and spikelet specification is delayed, resulting in prolonged branch formation and increased numbers of spikelets. In contrast, reductions in TAWAWA1 (TAW1) activity cause precocious IM abortion and spikelet formation, resulting in the generation of small inflorescences. TAW1 encodes a nuclear protein of unknown function and shows high levels of expression in the shoot apical meristem, the IM, and the BMs. TAW1 expression disappears from incipient spikelet meristems (SMs). We also demonstrate that members of the SHORT VEGETATIVE PHASE subfamily of MADS-box genes function downstream of TAW1. We thus propose that TAW1 is a unique regulator of meristem activity in rice and regulates inflorescence development through the promotion of IM activity and suppression of the phase change to SM identity.
Assuntos
Regulação da Expressão Gênica de Plantas/fisiologia , Inflorescência/anatomia & histologia , Meristema/crescimento & desenvolvimento , Oryza/fisiologia , Proteínas de Plantas/metabolismo , Elementos de DNA Transponíveis/genética , Fluorescência , Perfilação da Expressão Gênica , Hibridização In Situ , Inflorescência/metabolismo , Proteínas de Domínio MADS/metabolismo , Meristema/metabolismo , Proteínas Nucleares/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Cucurbita moschata FLOWERING LOCUS T-LIKE 2 (hereafter FTL2) and Arabidopsis thaliana (Arabidopsis) FLOWERING LOCUS T (FT), components of the plant florigenic signaling system, move long-distance through the phloem from source leaves to the vegetative apex where they mediate floral induction. The mechanisms involved in long-distance trafficking of FT/FTL2 remain to be elucidated. In this study, we identified the critical motifs on both FT and FTL2 required for cell-to-cell trafficking through mutant analyses using a zucchini yellow mosaic virus expression vector. Western blot analysis, performed on phloem sap collected from just beneath the vegetative apex of C. moschata plants, established that all mutant proteins tested retained the ability to enter the phloem translocation stream. However, immunolocalization studies revealed that a number of these FTL2/FT mutants were defective in the post-phloem zone, suggesting that a regulation mechanism for FT trafficking exists in the post-phloem unloading step. The selective movements of FT/FTL2 were further observed by microinjection and trichome rescue studies, which revealed that FT/FTL2 has the ability to dilate plasmodesmata microchannels during the process of cell-to-cell trafficking, and various mutants were compromised in their capacity to traffic through plasmodesmata. Based on these findings, a model is presented to account for the mechanism by which FT/FTL2 enters the phloem translocation stream and subsequently exits the phloem and enters the apical tissue, where it initiates the vegetative to floral transition.
Assuntos
Proteínas de Arabidopsis/metabolismo , Cucurbita/metabolismo , Floema/metabolismo , Proteínas de Plantas/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Arabidopsis/genética , Parede Celular/metabolismo , Sequência Conservada , Mutação , Floema/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Plasmodesmos/metabolismo , Transporte ProteicoRESUMO
Adventitious organogenesis in plant tissue culture involves de novo formation of apical meristems and should therefore provide important information about the fundamentals of meristem gene networks. We identified novel factors required for neoformation of the shoot apical meristem (SAM) through an analysis of shoot regeneration in root initiation defective3 (rid3) and root growth defective3 (rgd3) temperature-sensitive mutants of Arabidopsis. After induction of callus to regenerate shoots, cell division soon ceased and was then reactivated locally in the surface region, resulting in formation of mounds of dense cells in which adventitious-bud SAMs were eventually constructed. The rgd3 mutation inhibited reactivation of cell division and suppressed expression of CUP-SHAPED COTYLEDON1 (CUC1), CUC2 and SHOOT MERISTEMLESS (STM). In contrast, the rid3 mutation caused excess ill-controlled cell division on the callus surface. This was intimately related to enhanced and broadened expression of CUC1. Positional cloning revealed that the RGD3 and RID3 genes encode BTAF1 (a kind of TATA-binding protein-associated factor) and an uncharacterized WD-40 repeat protein, respectively. In the early stages of shoot regeneration, RGD3 was expressed (as was CUC1) in the developing cell mounds, whereas RID3 was expressed outside the cell mounds. When RID3 was over-expressed artificially, the expression levels of CUC1 and STM were significantly reduced. Taken together, these findings show that both negative regulation by RID3 and positive regulation by RGD3 of the CUC-STM pathway participate in proper control of cell division as a prerequisite for SAM neoformation.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Meristema/metabolismo , Sequência de Aminoácidos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proliferação de Células , Mapeamento Cromossômico , Clonagem Molecular , DNA de Plantas/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Teste de Complementação Genética , Meristema/genética , Meristema/crescimento & desenvolvimento , Dados de Sequência Molecular , Mutação , Análise de Sequência de DNA , TemperaturaRESUMO
Long-distance signaling via phloem tissues is an important mechanism for inter-organ communication. Such communication allows plants to integrate environmental information into physiological and developmental responses. Grafting has provided persuasive evidence of long-distance signaling involved in various processes, including flowering, tuberization, nodulation, shoot branching, post-transcriptional gene silencing, and disease resistance. A micro-grafting technique to generate two-shoot grafts is available for young seedlings of Arabidopsis thaliana and was adapted for use in the study of flowering. Histological analysis using transgenic plants expressing beta-glucuronidase (GUS) in phloem tissues showed that phloem continuity between a stock and a scion was established between 7 and 10 days after grafting. Experiments using tracer dyes and enhanced green fluorescent protein (EGFP) showed that the phloem connection was functional and capable of effecting macromolecular transmission. Successful grafts can be obtained at high frequency (10-30%) and selected after 2-3 weeks of post-surgery growth. This method was applied successfully to the study of flowering, one of the important events regulated by long-distance signaling. This grafting technique will facilitate the study of the long-distance action of genes involved in various aspects of growth and development, and in transport of signal molecules.
Assuntos
Arabidopsis/metabolismo , Flores/crescimento & desenvolvimento , Plântula/metabolismo , Transdução de Sinais , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Genes de Plantas , Hipocótilo/fisiologiaRESUMO
Day length perceived by a leaf is a major environmental factor that controls the timing of flowering. It has been believed that a mobile, long-distance signal called florigen is produced in the leaf under inductive day length conditions, and is transported to the shoot apex where it triggers floral morphogenesis. Grafting experiments have shown that florigen is transmissible from a donor plant that has been subjected to inductive day length to an uninduced recipient plant. However, the nature of florigen has long remained elusive. Arabidopsis FLOWERING LOCUS T (FT) is expressed in cotyledons and leaves in response to inductive long days (LDs). FT protein, with a basic region/leucine zipper (bZIP) transcription factor FD, acts in the shoot apex to induce target meristem identity genes such as APETALA1 (AP1) and initiates floral morphogenesis. Recent studies have provided evidence that the FT protein in Arabidopsis and corresponding proteins in other species are an important part of florigen. Our work shows that the FT activity, either from overexpressing or inducible transgenes or from the endogenous gene, to promote flowering is transmissible through a graft junction, and that an FT protein with a T7 tag is transported from a donor scion to the apical region of recipient stock plants and becomes detectable within a day or two. The sequence and structure of mRNA are not of critical importance for the long-distance action of the FT gene. These observations led to the conclusion that the FT protein, but not mRNA, is the essential component of florigen.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Reguladores de Crescimento de Plantas/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Sequência de Bases , Flores/genética , Genes de Plantas , Meristema/genética , Meristema/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta , Fotoperíodo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plasmídeos , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Transformação GenéticaRESUMO
Day length perceived by a leaf is a major environmental factor that controls the timing of flowering. It has been believed that a mobile, long-distance signal called florigen is produced in the leaf, and is transported to the shoot apex where it triggers floral morphogenesis. Grafting experiments have shown that florigen is transmissible from a donor plant that has been subjected to inductive day length to an un-induced recipient plant. However, the nature of florigen has long remained elusive. Recent studies have provided evidence that the FLOWERING LOCUS T (FT) protein in Arabidopsis and corresponding proteins in other species are an important part of florigen. Our work showed that the FT activity, either from overexpressing or inducible transgenes or from the endogenous gene, to promote flowering is transmissible through a graft junction, and that an FT protein with a T7 tag (FT-T7) is transported from a donor scion to the apical region of recipient stock plants and becomes detectable within a short period of 24-48 h. That the FT-green fluorescent protein (GFP) fusion protein (FT:GFP) retains limited ability for graft-transmissible action was confirmed.
RESUMO
FLOWERING LOCUS T (FT) is a conserved promoter of flowering that acts downstream of various regulatory pathways, including one that mediates photoperiodic induction through CONSTANS (CO), and is expressed in the vasculature of cotyledons and leaves. A bZIP transcription factor, FD, preferentially expressed in the shoot apex is required for FT to promote flowering. FD and FT are interdependent partners through protein interaction and act at the shoot apex to promote floral transition and to initiate floral development through transcriptional activation of a floral meristem identity gene, APETALA1 (AP1). FT may represent a long-distance signal in flowering.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Flores/crescimento & desenvolvimento , Transdução de Sinais , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Núcleo Celular/metabolismo , Cotilédone/metabolismo , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Domínio MADS , Meristema/genética , Meristema/metabolismo , Morfogênese , Mutação , Fenótipo , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Brotos de Planta/metabolismo , Mapeamento de Interação de Proteínas , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/genética , Ativação TranscricionalRESUMO
In Arabidopsis, shoots regenerate on calli derived from hypocotyl explants. Mutations in CUC1 and CUC2 (CUP-SHAPED COTYLEDON) reduce the induction of adventitious shoots on calli. To elucidate the function of CUC1 and CUC2 during this process, these genes were overexpressed in calli. Our results indicate that CUC1 and CUC2 promote adventitious shoot formation on calli. To clarify their functions, the concentrations of auxin and cytokinin in the shoot-inducing medium were changed. Calli of the single and double mutants of cuc1 and cuc2, as well as calli overexpressing either of the CUC genes, responded similarly. This suggests that neither of the genes are involved in synthesis or sensitivity of these hormones. During embryogenesis, CUC1 and CUC2 induce shoot apical meristem formation through activation of STM (SHOOT MERISTEMLESS). Our analyses using the stm mutant and an STM::GUS construct suggest that CUC1 and CUC2 also function upstream of STM even in calli.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Brotos de Planta/genética , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/fisiologia , Técnicas de Cultura , Citocininas/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/fisiologia , Ácidos Indolacéticos/farmacologia , Mutação , Reguladores de Crescimento de Plantas/farmacologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/crescimento & desenvolvimentoRESUMO
In higher plants, although several genes involved in shoot apical meristem (SAM) formation and organ separation have been isolated, the molecular mechanisms by which they function are largely unknown. CUP-SHAPED COTYLEDON (CUC) 1 and CUC2 are examples of two such genes that encode the NAC domain proteins. This study investigated the molecular basis for their activities. Nuclear localization assays indicated that green fluorescent protein (GFP)-CUC proteins accumulate in the nucleus. Yeast one-hybrid and transient expression assays demonstrated that the C-terminal domain (CTD) of the CUC has transactivation activity. Domain-swapping experiments revealed that the functional specificity of the CUC for promoting adventitious shoot formation resides in the highly conserved NAC domain, not in the CTD in which motifs specific to the CUC subfamily are located. Taken together, these observations suggest that CUC proteins transactivate the target genes involved in SAM formation and organ separation through a specific interaction between the NAC domain and the promoter region of the target genes.