A time-resolved model for the Arabidopsis shoot apical meristem during floral transition

Background. Aerial parts of plants originate from pluripotent cells in the shoot apical meristem (SAM). An important determinant of crop yield but also of importance to wild species in a natural environment is the correct timing of developmental transitions. This includes the differentiation from a vegetative into a reproductive SAM and the shutting down of developmental programs that build leaves, while inducing those that control flower formation. SAM size is dynamic and undergoes massive reorganization particularly throughout huge energy-consuming developmental changes such as floral transition. The presently unknown mechanisms controlling this process coincide with increased carbon (C) levels, for which the SAM is a sink. The Wahl group (The James Hutton Institute, JHI) has discovered dynamic feedback-regulations that ensure speeding up of cell cycle activity and metabolism at floral transition, and provide the basis and energy necessary to expand the SAM. This project seeks to incorporate known components of the flowering time and meristem maintenance networks with C signalling and integrate the dynamic changes throughout floral transition thereby further extending current models of SAM regulation generated in the Jönsson lab (Sainsbury Laboratory at University of Cambridge, SLCU).

Aims. Eventually, the project aims at generating an integral part for a revised, interactive SAM regulatory model enabling the community to formulate testable hypotheses for stimuli-response relationships and pave the way to address new questions beyond the framework of this project. The combination of cutting-edge and well-established methods will provide novel insights into the mechanisms underlying the dramatic morphological changes at the SAM during floral transition. The core objectives are: (i) the morphological, transcriptional and metabolic monitoring of SAM dynamics, (ii) the analysis of spatial dynamics of important regulators in response to C availability at the SAM throughout floral transition, (iii) the computational analysis of the data to produce time-resolved models.

Methods/Approach. We will make use of a unique combination and innovative application of established methodologies such as three-dimensional high-resolution microscopy, metabolite analyses, and transcript (RT-qPCR, RNA in situ hybridization) and transcriptome analyses (RNA-seq). FRET biosensors combined with fluorophore-tagged marker gene expression will provide spatiotemporal resolution of C levels and gene expression at the SAM. Via a computational approach we will incorporate the data into existing morphological models for meristem maintenance, to formulate testable hypotheses for stimuli-response relationships.

Outline for the 1^st 12-18 months. The student will familiarize themself with the diverse techniques including the plant growth regimes required for this project. A collection of available mutant and transgenic lines for central components of C-signalling, as well as of the meristem maintenance and flowering time networks will be initially tested when grown in these growth regimes. Using confocal microscopy, we will first approach the dynamics of the SAM throughout floral transition by observation of morphology in a time series spanning floral transition (vegetative, transition, reproductive SAM). For in vivo analyses of sucrose concentrations, we will make use of a series of new sucrose FRET biosensors with an expanded detection range from micro- to millimolar sucrose concentrations. A small set of available fluorophore-tagged markers will be used for spatiotemporal resolution in live cells in plants.

At a later stage, computer algorithms for the live-imaging data will result in 3D-datasets, which can be used to generate 3D-models suitable to not only visualize structures, but also to calculate volumes and their dynamic changes during floral transition. The time-resolved effects of C signalling and availability will be determined using RNA-seq on whole SAMs.

Opportunity for intellectual input. Following initial characterization and computational analysis of SAM dynamics at floral transition, the direction of the research will be determined by the student’s interests in consultation with the supervisors. A number of potential avenues exist including deeper understanding of the signalling pathways influencing C-dependent floral transition. Since the computational approaches will result in testable hypotheses, the student can use the experience gained to follow up leads in ‘proof of concept’ experiments, e.g. by testing mutant lines for candidate genes (T-DNA insertion, CRISPR/Cas9, or SAM specific artificial microRNA expression) through monitoring phenotypic, transcriptional and metabolic changes.