RESUMO
How biological systems generate reproducible patterns with high precision is a central question in science. The shoot apical meristem (SAM), a specialized tissue producing plant aerial organs, is a developmental system of choice to address this question. Organs are periodically initiated at the SAM at specific spatial positions and this spatiotemporal pattern defines phyllotaxis. Accumulation of the plant hormone auxin triggers organ initiation, whereas auxin depletion around organs generates inhibitory fields that are thought to be sufficient to maintain these patterns and their dynamics. Here we show that another type of hormone-based inhibitory fields, generated directly downstream of auxin by intercellular movement of the cytokinin signalling inhibitor ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEIN 6 (AHP6), is involved in regulating phyllotactic patterns. We demonstrate that AHP6-based fields establish patterns of cytokinin signalling in the meristem that contribute to the robustness of phyllotaxis by imposing a temporal sequence on organ initiation. Our findings indicate that not one but two distinct hormone-based fields may be required for achieving temporal precision during formation of reiterative structures at the SAM, thus indicating an original mechanism for providing robustness to a dynamic developmental system.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Transporte Biológico , Citocininas/antagonistas & inibidores , Transdução de Sinais , Arabidopsis/anatomia & histologia , Arabidopsis/citologia , Citocininas/metabolismo , Ácidos Indolacéticos/metabolismo , Meristema/metabolismo , Reguladores de Crescimento de Plantas/antagonistas & inibidores , Reguladores de Crescimento de Plantas/metabolismo , Brotos de Planta/metabolismoRESUMO
Cell differentiation has been associated with changes in mechanical stiffness in single-cell systems, yet it is unknown whether this association remains true in a multicellular context, particularly in developing tissues. In order to address such questions, we have developed a methodology, termed quantitative tandem epifluorescence and nanoindentation, wherein we sequentially determine cellular genetic identity with confocal microscopy and mechanical properties with atomic force microscopy. We have applied this approach to examine cellular stiffness at the shoot apices of Arabidopsis (Arabidopsis thaliana) plants carrying a fluorescent reporter for the CLAVATA3 (CLV3) gene, which encodes a secreted glycopeptide involved in the regulation of the centrally located stem cell zone in inflorescence and floral meristems. We found that these CLV3-expressing cells are characterized by an enhanced stiffness. Additionally, by tracking cells in young flowers before and after the onset of GREEN FLUORESCENT PROTEIN expression, we observed that an increase in stiffness coincides with this onset. This work illustrates how quantitative tandem epifluorescence and nanoindentation can reveal the spatial and temporal dynamics of both gene expression and cell mechanics at the shoot apex and, by extension, in the epidermis of any thick tissue.
RESUMO
The plant hormone auxin is thought to provide positional information for patterning during development. It is still unclear, however, precisely how auxin is distributed across tissues and how the hormone is sensed in space and time. The control of gene expression in response to auxin involves a complex network of over 50 potentially interacting transcriptional activators and repressors, the auxin response factors (ARFs) and Aux/IAAs. Here, we perform a large-scale analysis of the Aux/IAA-ARF pathway in the shoot apex of Arabidopsis, where dynamic auxin-based patterning controls organogenesis. A comprehensive expression map and full interactome uncovered an unexpectedly simple distribution and structure of this pathway in the shoot apex. A mathematical model of the Aux/IAA-ARF network predicted a strong buffering capacity along with spatial differences in auxin sensitivity. We then tested and confirmed these predictions using a novel auxin signalling sensor that reports input into the signalling pathway, in conjunction with the published DR5 transcriptional output reporter. Our results provide evidence that the auxin signalling network is essential to create robust patterns at the shoot apex.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Ácidos Indolacéticos/farmacologia , Reguladores de Crescimento de Plantas/farmacologia , Brotos de Planta/crescimento & desenvolvimento , Transdução de Sinais/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Análise por Conglomerados , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Hibridização in Situ Fluorescente , Meristema/química , Meristema/metabolismo , Microscopia Confocal , Modelos Teóricos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Organogênese , Brotos de Planta/genética , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas , Transcrição GênicaRESUMO
To control morphogenesis, molecular regulatory networks have to interfere with the mechanical properties of the individual cells of developing organs and tissues, but how this is achieved is not well known. We study this issue here in the shoot meristem of higher plants, a group of undifferentiated cells where complex changes in growth rates and directions lead to the continuous formation of new organs. Here, we show that the plant hormone auxin plays an important role in this process via a dual, local effect on the extracellular matrix, the cell wall, which determines cell shape. Our study reveals that auxin not only causes a limited reduction in wall stiffness but also directly interferes with wall anisotropy via the regulation of cortical microtubule dynamics. We further show that to induce growth isotropy and organ outgrowth, auxin somehow interferes with the cortical microtubule-ordering activity of a network of proteins, including AUXIN BINDING PROTEIN 1 and KATANIN 1. Numerical simulations further indicate that the induced isotropy is sufficient to amplify the effects of the relatively minor changes in wall stiffness to promote organogenesis and the establishment of new growth axes in a robust manner.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Ácidos Indolacéticos/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/metabolismo , Fenômenos Biomecânicos , Parede Celular/metabolismo , Katanina , Meristema/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Brotos de Planta/crescimento & desenvolvimento , Receptores de Superfície Celular/metabolismoRESUMO
A functional study of mutants of the human immunodeficiency virus type 1 (HIV-1) integrase (IN) was conducted with the support of a recently proposed HIV-1 intasome model. Firstly, we investigated the predicted position of the C-terminal domain (CTD) and the flexibility of the alpha-6 helix by mutating the residue Ile-203. This had no impact on the 3'-processing reaction but reduced the strand transfer reaction and the formation of tetramers. Secondly, the residues Ile-141 of the catalytic loop and Glu-246 of the CTD are predicted to bind the Td-3 base of the viral DNA maintaining it in a "flipped out" position and stabilizing the catalytic core domain (CCD)-CTD interface. Our data showed that the Ile-141/Td-3 interaction was important for the strand transfer activity and the oligomerization of IN. Interestingly, mutating the Glu-246 residue by an alanine enhanced half- and full-site integrations, suggesting that this residue may not be optimized for integration.