Final Report Summary - PLANT-DEVELOPMENT (Investigating a role for the plant hormone, auxin, in coordinating dorsiventral boundary establishment during organogenesis in Arabidopsis thaliana)
The aim of this proposal is to extend our understanding of how two important classes of plant-specific patterning genes, the class III homeodomain-leucine zipper (HD-ZIP III) and Kanadi (KAN) transcription factor families, act together with the plant hormone, auxin, to establish boundaries and promote new axes of growth during organ development. By combining a sophisticated live multi-spectral confocal imaging approach with novel perturbation strategies, we aim to investigate how two key patterning mechanisms, PIN-mediated auxin flow and the expression of the HD-ZIP III and KAN polarity genes, influence one another during dorsiventral boundary establishment.
An important finding coming from this study is that mosaic expression of a fluorescently-tagged microRNA-resistant HD-ZIP III transcription factor (REV*-YPET) is sufficient to induce outgrowths of tissue (i.e. laminar outgrowths) on the underside of leaves. We have demonstrated that mosaic expression of REV*-YPET, results in striking laminar outgrowths on the ventral (under) side of leaves and other leaf-like organs, such as sepals and petals. This is significant as it demonstrates that the dorsiventral boundary is sufficient to produce new directions of growth. By varying the length of the heat shock treatment, and hence the spatial domain of REV*-YPET genetic sectors, it was possible to obtain a variety of leaf phenotypes, ranging from small patches of ectopic lamina to trumpet shaped leaves and even fully radialised organs. We found that developmental context was important. Beyond a certain developmental window, ectopic REV*-YPET expression did not result in outgrowths.
The ambitious experiments outlined in this project proposal required the development of novel genetic tools. We have invested significant effort towards developing a customised transgenic platform for generating mosaic plants, which is compatible with live imaging approaches. Part of the challenge was to obtain plants with up to six transgenes in a timely manner. The resulting sectoring system exploits a heat-shock inducible Cre/loxP recombination system and gateway recombination technology to allow any gene of interest to be 'knocked-in' and tracked using a minimum of four gene cassettes. These four gene cassettes are combined into two separate transgenes and introduced into fluorescent marker lines to facilitate a live-imaging approach. We currently have eight unique genes cloned into this new sectoring system (REV*-YPET, KAN1, YAB3, mir165, ZPR3, WOX1, iaaL, YUCCA4). Preliminary observations of plants with mosaic expression of these genes of interest reveal various ectopic outgrowths and changes in leaf morphology. These exciting changes in morphology warrant further detailed investigation.
The improved CRE/loxP sectoring system allows us to dynamically follow changes in gene patterning at newly forming dorsiventral boundaries using live multi-spectral confocal imaging. Due to technical difficulties imaging young leaf primordia we resorted to imaging of other leaf like organs, e.g. sepals, following mosaic knock-in of REV*-YPET. Initial observations of KAN1 expression (from a pKAN1:KAN1-2GFP reporter gene) in the sepals of sectored plants revealed that induction of REV*-YPET can lead to repression of KAN1 gene expression. This is a significant finding as it provides a hint as to how ectopic REV*-YPET expression could result in directional tissue outgrowth. Further detailed studies are required to fully appreciate the genetic mechanism underlying this phenomenon.
In addition to the above sectoring experiments, we have performed time-lapse live-imaging of kan triple (k123) mutant seedlings expressing two reporter genes: pPIN1:PIN1-GFP and pREV:REV-2VENUS. These experiments revealed that ectopic outgrowths observed on the ventral side of mutant leaves coincide with the ectopic REV expression boundary, providing further support for a model in which directional tissue growth occurs at the dorsiventral boundary. Progress has also been made towards developing new methods for following early leaf development using live confocal imaging.
Further work is required to fully elucidate the genetic mechanisms underlying dorsiventral boundary formation and laminar outgrowth. The genetic tools generated during this project along with the knowledge gained about ectopic outgrowths will be invaluable for following changes in reporter gene expression during dorsiventral boundary formation and tissue outgrowth using live-imaging. This is an ongoing project.
An important finding coming from this study is that mosaic expression of a fluorescently-tagged microRNA-resistant HD-ZIP III transcription factor (REV*-YPET) is sufficient to induce outgrowths of tissue (i.e. laminar outgrowths) on the underside of leaves. We have demonstrated that mosaic expression of REV*-YPET, results in striking laminar outgrowths on the ventral (under) side of leaves and other leaf-like organs, such as sepals and petals. This is significant as it demonstrates that the dorsiventral boundary is sufficient to produce new directions of growth. By varying the length of the heat shock treatment, and hence the spatial domain of REV*-YPET genetic sectors, it was possible to obtain a variety of leaf phenotypes, ranging from small patches of ectopic lamina to trumpet shaped leaves and even fully radialised organs. We found that developmental context was important. Beyond a certain developmental window, ectopic REV*-YPET expression did not result in outgrowths.
The ambitious experiments outlined in this project proposal required the development of novel genetic tools. We have invested significant effort towards developing a customised transgenic platform for generating mosaic plants, which is compatible with live imaging approaches. Part of the challenge was to obtain plants with up to six transgenes in a timely manner. The resulting sectoring system exploits a heat-shock inducible Cre/loxP recombination system and gateway recombination technology to allow any gene of interest to be 'knocked-in' and tracked using a minimum of four gene cassettes. These four gene cassettes are combined into two separate transgenes and introduced into fluorescent marker lines to facilitate a live-imaging approach. We currently have eight unique genes cloned into this new sectoring system (REV*-YPET, KAN1, YAB3, mir165, ZPR3, WOX1, iaaL, YUCCA4). Preliminary observations of plants with mosaic expression of these genes of interest reveal various ectopic outgrowths and changes in leaf morphology. These exciting changes in morphology warrant further detailed investigation.
The improved CRE/loxP sectoring system allows us to dynamically follow changes in gene patterning at newly forming dorsiventral boundaries using live multi-spectral confocal imaging. Due to technical difficulties imaging young leaf primordia we resorted to imaging of other leaf like organs, e.g. sepals, following mosaic knock-in of REV*-YPET. Initial observations of KAN1 expression (from a pKAN1:KAN1-2GFP reporter gene) in the sepals of sectored plants revealed that induction of REV*-YPET can lead to repression of KAN1 gene expression. This is a significant finding as it provides a hint as to how ectopic REV*-YPET expression could result in directional tissue outgrowth. Further detailed studies are required to fully appreciate the genetic mechanism underlying this phenomenon.
In addition to the above sectoring experiments, we have performed time-lapse live-imaging of kan triple (k123) mutant seedlings expressing two reporter genes: pPIN1:PIN1-GFP and pREV:REV-2VENUS. These experiments revealed that ectopic outgrowths observed on the ventral side of mutant leaves coincide with the ectopic REV expression boundary, providing further support for a model in which directional tissue growth occurs at the dorsiventral boundary. Progress has also been made towards developing new methods for following early leaf development using live confocal imaging.
Further work is required to fully elucidate the genetic mechanisms underlying dorsiventral boundary formation and laminar outgrowth. The genetic tools generated during this project along with the knowledge gained about ectopic outgrowths will be invaluable for following changes in reporter gene expression during dorsiventral boundary formation and tissue outgrowth using live-imaging. This is an ongoing project.