Combining genetic and modelling approaches to understanding vascular development in Arabidopsis
Lead Research Organisation:
Durham University
Department Name: Biosciences
Abstract
Background: In higher plants, post-embryonic tissues are derived from pluripotent stem cell populations present in niches referred to as meristems. Meristems are composed of central organizing cells with adjacent dividing stem cells that provide cells for new organs. Meristems that are responsible for length-wise plant growth are located at the root and shoot apices. Radial expansion also occurs via divisions in meristematic tissues, referred to as the cambium and procambium, which are present in the vascular tissue. Plant vascular tissue is constituted of two conductive tissues, the xylem and phloem. Xylem is required for water transport, and phloem is required primarily for nutrient transport. A significant proportion of terrestrial biomass is constituted of xylem cells that make up woody plant tissue, which represents a large renewable resource of energy and biomaterials.
Both xylem and phloem are derived from cell divisions in vascular meristems known as the cambium and procambium. One major component that influences several aspects of plant vascular development, is a signalling module characterised by a peptide ligand called TDIF that is expressed in the phloem, and its cognate receptor, PXY which is procambium expressed. The non-cell autonomous interaction of these factors results in particularly complex outputs. This ligand-receptor pair is thought to regulate the rate of cell division in the procambium, exclude xylem identity from the division zone and also control vascular organization by influencing the orientation of cell division. TDIF influences these outputs via interactions with a number of other signalling components and transcriptional regulators. While the identification of targets and interactors that regulate various aspects of PXY signalling outputs has been successful, it remains unclear as to how these complex outputs are integrated. Aims: In order to understand how the three outputs of PXY signalling (i.e. vascular organisation, cell division and exclusion of xylem identity from the procambium) are integrated, we aim to generate quantitative data that will be used to make a logical network model that simulates PXY signalling. Our model will assist in generating hypotheses that will be tested using genetics and molecular biology.
Methodology: The student will initially combine the use of histology, microscopy and imaging methodologies with computational analysis (such as cell profiler software) to determine precise numbers of xylem phloem and procambium cells present in vascular tissue during inflorescence development. These analyses will be combined with gene expression data that the student will generate using quantitative RT-PCR. Wild-type Arabidopsis and pxy mutants will be used, as will lines where TDIF has been removed. TDIF is derived from 3 genes, and we have collaborated with Zack Nimchuk (University of North Carolina) who has generated a triple mutant using CRISPR/Cas9 to entirely eliminate the ligand. This data will be used build a mathematical/computational model of the system. The model will take the form of a spatially distributed logical network, generated by the student under the supervision of co-supervisor Natasha Savage (University of Liverpool). This model will then be used to produce testable hypotheses.
Subsequent experiments will be guided by modelling data. We envisage model predictions will be tested by manipulating gene expression in planta. For example, if the model predicts that negative feedback of components is critical for maintaining the system, we will manipulate expression using heterologous and/or inducible promoters that are not susceptible to such feedback and assessing the consequences for vascular development. Other manipulations could be used to test a variety of emergent properties including morphogen gradients, rates of cell division etc.
Timetable of Activities:
Month 0-12: Generate growth data and gene expression data for Arabidopsis vascular tissu
Both xylem and phloem are derived from cell divisions in vascular meristems known as the cambium and procambium. One major component that influences several aspects of plant vascular development, is a signalling module characterised by a peptide ligand called TDIF that is expressed in the phloem, and its cognate receptor, PXY which is procambium expressed. The non-cell autonomous interaction of these factors results in particularly complex outputs. This ligand-receptor pair is thought to regulate the rate of cell division in the procambium, exclude xylem identity from the division zone and also control vascular organization by influencing the orientation of cell division. TDIF influences these outputs via interactions with a number of other signalling components and transcriptional regulators. While the identification of targets and interactors that regulate various aspects of PXY signalling outputs has been successful, it remains unclear as to how these complex outputs are integrated. Aims: In order to understand how the three outputs of PXY signalling (i.e. vascular organisation, cell division and exclusion of xylem identity from the procambium) are integrated, we aim to generate quantitative data that will be used to make a logical network model that simulates PXY signalling. Our model will assist in generating hypotheses that will be tested using genetics and molecular biology.
Methodology: The student will initially combine the use of histology, microscopy and imaging methodologies with computational analysis (such as cell profiler software) to determine precise numbers of xylem phloem and procambium cells present in vascular tissue during inflorescence development. These analyses will be combined with gene expression data that the student will generate using quantitative RT-PCR. Wild-type Arabidopsis and pxy mutants will be used, as will lines where TDIF has been removed. TDIF is derived from 3 genes, and we have collaborated with Zack Nimchuk (University of North Carolina) who has generated a triple mutant using CRISPR/Cas9 to entirely eliminate the ligand. This data will be used build a mathematical/computational model of the system. The model will take the form of a spatially distributed logical network, generated by the student under the supervision of co-supervisor Natasha Savage (University of Liverpool). This model will then be used to produce testable hypotheses.
Subsequent experiments will be guided by modelling data. We envisage model predictions will be tested by manipulating gene expression in planta. For example, if the model predicts that negative feedback of components is critical for maintaining the system, we will manipulate expression using heterologous and/or inducible promoters that are not susceptible to such feedback and assessing the consequences for vascular development. Other manipulations could be used to test a variety of emergent properties including morphogen gradients, rates of cell division etc.
Timetable of Activities:
Month 0-12: Generate growth data and gene expression data for Arabidopsis vascular tissu
Organisations
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/M011186/1 | 01/10/2015 | 31/03/2024 | |||
| 1786071 | Studentship | BB/M011186/1 | 01/10/2016 | 31/03/2021 | Kristine Bagdassarian |
| Description | Vascular tissues develop in a highly organised manner, with different cell types arranged in concentric rings. The two main types of transporting tissues, the xylem (which transports water) and the phloem (which transports nutrients) are derived on either side of the meristematic cambium. The mechanisms coordinating the balance between cell division and differentiation to create this pattern are not fully understood. An objective to this research is to elucidate the genetic interactions underlying these processes. Two receptor-ligand pairs have been implicated in governing vascular development, namely the ERECTA (ER) - EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) and the PHLOEM INTERCALLATED WITH XYLEM (PXY) - TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF). ER was shown to interact with PXY, which is expressed in the procambium but perceives the TDIF, whose gene CLE41 is expressed in the phloem. Thus, PXY-TDIF represent communication between the different tissue types. In this research, we have established that ER regulates expression of PXY paralogues, PXL1 and PXL2, and that in turn PXY, PXL1 and PXL2 (collectively PXf) together with ER, regulate the expression of ERL1 and ERL2, genes paralogous to ER (collectively ERf). To determine how genetic interactions between the PXf and ERf might control cell fate in plant vascular tissue, we used Arabidopsis hypocotyl radial growth as a model system. pxy pxl1 pxl2 er erl1 erl2 lines were generated using a combination of classical genetics and genome editing. The radius of the hypocotyls was similar in wild type and pxf er mutants, but smaller in the absence of further ER family genes. A MATLAB code was written to determine the morphological changes caused by the loss of erf and pxf at the cellular level. Xylem cell size was higher in pxf, pxf er and pxf er erl1 lines than wild type, but not in pxf er erl2 or pxf erf lines. Thus the erl2 gene is critical in increasing cell size to compensate for the reduction in cell divisions in pxf mutants. Our results demonstrate that PXY and ER gene families genetically interact to coordinate vascular division and cell morphology to maintain radial growth in Arabidopsis thaliana hypocotyl vascular tissue. |
| Exploitation Route | The ER family of genes is not well studied in the context of hypocotyls and only slightly better investigated in the context of other plant organs. To better understand the mechanisms driving vascular development, it's essential to study the ER family and its relationship to PXY in further detail. The MATLAB methodology applied in the research can be used in other fields, with interest being shown by the Durham engineering department for their own research purposes. |
| Sectors | Agriculture, Food and Drink |
| URL | https://dev.biologists.org/content/146/10/dev177105 |
| Title | MATLAB Algorithm for Analysing Cell Morphology |
| Description | To capture measurements for the cell perimeters and areas between the different genotypes analysed in our research 'Paralogues of the PXY and ER receptor kinases enforce radial patterning in plant vascular tissue.', images from 6 different individuals were selected for each genotype tested. Minimums of 10 cells of each cell type (xylem vessels, xylem fibres, phloem and parenchyma) were selected from a wedge with a 60-degree central angle from each image. Cells of each type were selected along the full length of the radial axis to ensure that cells of all sizes and phenotypic variation were represented. A MATLAB code was generated to extract the intrinsic properties of each cell type. To that end, the code was designed to split each image into binary sub-images, wherein the interior of the cell type of interest was represented as white objects on black background. The cells (the white objects) from each image were then analysed as connected components of the image and their area and perimeter extracted. To remove noise, i.e. data obtained from objects which were wrongly classified as connected components within the algorithm (e.g. stray pixels), the code was devised to discard data which yielded unrealistically small values for perimeter and area (perimeter value of 0 µM, area smaller than 1 µM). The data was converted from pixels to microns using a calibration factor, in order to yield results consistent with laboratory observations. For each cell type, an equal number of cells was selected on a random basis from each plant within each genotype to avoid small variations between the number of representatives obtained from each individual plant. To test the significance of the variation between the cell areas and perimeters between the different genotypes, a nested ANOVA was performed in R at 5% significance level. To perform the nested ANOVA, the data was classified according to genotype (treatment) and plant ID (plants within that treatment), with the response variable either the area or perimeter. A post-hoc Tukey HSD test was performed to determine the significance of the pairwise differences between the means of the areas/perimeters between the different genotypes. Mean hypocotyl diameters were measured using callipers. Radius was calculated from hypocotyl images of six plants from each genotype. A MATLAB code was used to measure the length of the shorter radius. The length of the radii in pixels was subsequently converted to microns. A Lilliefors test at 5% significance level was used to confirm that the radii for each genotype were normally distributed. A one-way ANOVA followed by a post-hoc Tukey HSD test was used to determine pairwise variation between the means. |
| Type Of Material | Computer model/algorithm |
| Year Produced | 2019 |
| Provided To Others? | Yes |
| Impact | This is a good way to gain insight into the quantitative properties of images of plant cross sections or other similar objects in other areas. |
| URL | https://github.com/KristineBagdassarian/PXY-ER_enforce_radial_growth |