RESUMO
The regulatory mechanisms involved in plant development include many signals, some of them acting as graded positional cues regulating gene expression in a concentration-dependent manner. These regulatory molecules, that can be considered similar to animal morphogens, control cell behavior in developing organs. A suitable experimental approach to study expression gradients in plants is quantitative laser scanning confocal microscopy (LSCM) using Arabidopsis thaliana root tips as a model system. In this chapter, we outline a detailed method for image acquisition using LSCM, including detailed microscope settings and image analysis using FIJI as software platform.
Assuntos
Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Processamento de Imagem Assistida por Computador/métodos , Microscopia Confocal/métodos , Raízes de Plantas/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/ultraestrutura , SoftwareRESUMO
An increase in crop yield is essential to reassure food security to meet the accelerating global demand. Several genetic modifications can increase organ size, which in turn might boost crop yield. Still, only in a few cases their performance has been evaluated under stress conditions. MicroRNA miR396 repress the expression of GROWTH-REGULATING FACTOR (GRF) genes that codes for transcription factors that promote organ growth. Here, we show that both Arabidopsis thaliana At-GRF2 and At-GRF3 genes resistant to miR396 activity (rGRF2 and rGRF3) increased organ size, but only rGRF3 can produce this effect without causing morphological defects. Furthermore, introduction of At-rGRF3 in Brassica oleracea can increase organ size, and when At-rGRF3 homologs from soybean and rice are introduced in Arabidopsis, leaf size is also increased. This suggests that regulation of GRF3 activity by miR396 is important for organ growth in a broad range of species. Plants harboring rGRF3 have larger leaves also under drought stress, a condition that stimulates miR396 accumulation. These plants also showed an increase in the resistance to virulent bacteria, suggesting that the size increment promoted by rGRF3 occurs without an obvious cost on plant defenses. Our findings indicate that rGRF3 can increase plant organ size under both normal and stress conditions and is a valuable tool for biotechnological applications.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Folhas de Planta/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Brassica/genética , Brassica/crescimento & desenvolvimento , MicroRNAs/genética , MicroRNAs/metabolismo , Tamanho do Órgão/genética , Oryza/genética , Oryza/crescimento & desenvolvimento , Folhas de Planta/genética , RNA de Plantas/genética , RNA de Plantas/metabolismo , Glycine max/genética , Glycine max/crescimento & desenvolvimento , Fatores de Transcrição/genéticaRESUMO
In the root meristem, the quiescent center (QC) is surrounded by stem cells, which in turn generate the different cell types of the root. QC cells rarely divide under normal conditions but can replenish damaged stem cells. In the proximal meristem, the daughters of stem cells, which are referred to as transit-amplifying cells, undergo additional rounds of cell division prior to differentiation. Here, we describe the functions of GRF-INTERACTING FACTORs (GIFs), including ANGUSTIFOLIA3 (AN3), in Arabidopsis thaliana roots. GIFs have been shown to interact with GRF transcription factors and SWI/SNF chromatin remodeling complexes. We found that combinations of GIF mutants cause the loss of QC identity. However, despite their QC impairment, GIF mutants have a significantly enlarged root meristem with additional lateral root cap layers. We show that the increased expression of PLETHORA1 (PLT1) is at least partially responsible for the large root meristems of an3 mutants. Furthermore, we found that GIFs are necessary for maintaining the precise expression patterns of key developmental regulators and that AN3 complexes bind directly to the promoter regions of PLT1 as well as SCARECROW We propose that AN3/GIFs participate in different pathways that control QC organization and the size of the meristem.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Diferenciação Celular/genética , Divisão Celular/genética , Montagem e Desmontagem da Cromatina/genética , Homeostase/genética , Meristema/genética , Meristema/crescimento & desenvolvimento , Meristema/fisiologia , Mutação , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/fisiologia , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The combinatory effects of cell proliferation and cell elongation determines the rate at which organs growth. In the root meristematic zone cells both divide and expand, while post-mitotic cells in the elongation zone only expands until they reach their final size. The transcription factors of the GROWTH-REGULATING FACTOR (GRF) class promote cell proliferation in various plant organs. Their expression is restricted to cells with a high proliferative capacity, yet strong downregulation of the GRF activity compromise the plant survival. Part of expression pattern of the GRFs is ensured by the post-transcriptional repression mediated by the conserved microRNA miR396. Here we show the quantitative effects in root growth caused by GRF depletion in a series of transgenic lines with different miR396 levels. We show that high miRNA levels affect cell elongation and proliferation in roots. Detailed analysis suggests that cell proliferation is restricted due to a reduction in cell cycle speed that might result from defects in the accumulation of mitotic cyclins. The results provide insights into the participation of the miRNA-GRF regulatory network in root development.