Dynamics of chromosome organization during reproductive development – DYSCORD

Objective 1. Creation of multi-fluorophore ANCHOR collections.

Three pairs of ANCHOR systems (OR1–ANCH1, OR2–ANCH2, OR4–ANCH4) are available, but only OR2–ANCH2 has been so far validated in plants. Various molecular constructs containing the ANCH1, 2, or 4 sequence and the OR (1, 2, or 4) gene fused to a fluorescent protein under a constitutive promoter have been synthesized and transferred into plants. Fluorescence in root cell nuclei (uniform or focal, intensity) is analyzed by confocal microscopy in each ANCHOR line (T1 transformants).

Objective 2.

To study chromosome pairing during meiosis, an ANCHOR construct expressing fluorescent OR2 under a meiosis-specific promoter has been generated and introduced into plants. The robustness of the fluorescent signal in various ANCHOR lines is evaluated by time-lapse confocal microscopy on male meiocytes in anthers. In a subsequent step, the genomic location of the ANCHOR transgene will be determined.

Objective 3.

To track the dynamics of alleles of a gene and their transcriptional status, a DUAL molecular construct has been synthesized. It includes an ANCHOR module to monitor gene position and dynamics, and an MCP–MS2 module to image the corresponding mRNA expression. The intensity and localization of fluorescence emitted by ANCHOR and MCP–MS2 are evaluated in vegetative and reproductive tissues by confocal microscopy.

Objective 1.

Since the start of the project, 745 transgenic lines have been generated with the ANCHOR2 system (OR2–anch2), containing the ANCH2 sequence and the OR2 gene fused to a fluorescent protein (mScarlet, mStayGold) under a strong constitutive promoter. These lines represent markers at various genomic positions in Arabidopsis thaliana. Additionally, 19 lines expressing OR2–mStaygold under a meiosis-specific promoter were obtained.

For the other ANCHOR systems, a smaller collection of 42 lines has been generated using ANCHOR1 (OR1–anch1) fused to the green fluorescent protein mNeonGreen. However, this construct produced an undesired nucleolar background signal, requiring the creation of new molecular constructs using mStayGold instead.

Objective 2.

The first phase of the project focused on adapting the ANCHOR technology for analyzing chromosome pairing during meiosis. We have now validated a series of “meioANCH” lines in which ANCH loci can be visualized in meiocytes over long time periods (several hours). Using homozygous plants for the ANCH locus, we observed for the first time the pairing of homologous chromosomes in vivo in plant tissues. This approach allows us to distinguish between stages where the two homologous ANCH loci behave independently and those where they are closely associated.

Objective 3.

One goal of Work Package 3 is to simultaneously monitor the subnuclear dynamics of allelic variants of imprinted genes and their real-time transcriptional activity during endosperm development. Joint analysis of allele dynamics and transcription at an endogenous locus is currently not possible due to the low efficiency of targeted integration (knock-in) approaches.

As an alternative, we implemented a strategy based on a synthetic transgene called DUAL, which combines an ANCHOR tagging module for allele tracking and an mRNA imaging cassette using the MCP–MS2 system. A first pilot DUAL construct has been synthesized. The inclusion of 128x MS2 repeats caused plasmid instability, significantly delaying the generation of transgenic lines. Initial confocal microscopy observations in root cells revealed uniform fluorescence from MCP–mStayGold and foci of OR–mScarlet2x fluorescence. Despite using constitutive promoters and state-of-the-art fluorescent proteins, the fluorescence levels detected in endosperm nuclei remain insufficient for further analysis.

Objective 1.

The goal is to increase the number of ANCHOR1 lines to around 2000 and characterize the transgene genomic location for several hundred of them. The labeling capacity of the ANCHOR4 system (OR4–anch4) is currently being evaluated. In the long term, two focused collections of about 100 independent lines will be established for the ANCHOR1 and ANCHOR4 systems, respectively.

Objective 2.

Building on the already generated meioANCH lines, our work now focuses on transition phases to understand how homologous chromosomes first make contact in the nucleus and how these initial interactions become stabilized during meiosis.

Objective 3.

New DUAL constructs are being synthesized to test their functionality first in somatic cells.

Sexual reproduction in plants involves key developmental phases that first generate genetic distinct haploid spores during meiosis. After several rounds of division, the male and female spores eventually differentiate respectively into two male gametes (sperm cells) and two female gametes (egg cell, central cell). Double fertilization of the gametes then produces an embryo and its feeding tissue the endosperm that both develop inside a seed. During these phases, massive changes in cellular compartments, chromosome architecture and chromatin structure occur in a relatively short period of time. Unfortunately, these events take place deep inside the flower organs and in a limited number of cells, which complicates investigations and has left many questions unresolved for many years. Live imaging protocols have now been developed to observe reproductive cells during all key phases of plant reproduction (meiosis, gametogenesis, fertilization, embryogenesis) and greatly enhanced knowledge of some dynamic events. However, there is still a lack of tools to address any scientific question related to chromosome dynamics in living reproductive tissues. The recent development by partners of the project of the ANCHOR system - a new DNA-labelling system allowing live-cell imaging of a single locus in planta- provides an unprecedented possibility to fill the gap. ANCHOR comprises an OR protein fused to a fluorescent reporter that binds a ANCH target sequence, allowing robust live-cell imaging of a single locus in planta.
Taking advantage of state-of-the-art live-imaging approaches, The DYSCORD project aims to better understand two important reproduction steps: (i) parental chromosome recognition during meiosis and (ii) genomic imprinting in the endosperm. In work package 1, a collection of ANCHOR lines distributed throughout the Arabidopsis thaliana genome will be generated to allow the simultaneous observation of up to three unlinked single-copy loci in a single living nucleus. It should be noted that this collection will be accessible to the entire scientific community. In the second work package, we will study chromosome dynamics during meiosis, a step in which the parental chromosomes become tightly engaged one with another. We will use the ANCHOR lines to determine the dynamics of homologous chromosome interactions and compare the behaviour of different chromosomal regions either in wild type or in meiotic mutants. The work package 3 will focus on understanding how imprinted genes characterized by a monoallelic expression behave during endosperm development using live imaging. The dynamics of the distinct fluorescent ANCHOR-tagged parental alleles and their transcriptional activity will be simultaneously monitored using live RNA-based imaging technologies during endosperm development in wild type and in mutants that restore biallelic expression.
The results obtained in DYSCORD on A. thaliana will benefit the entire scientific community working in the field of meiosis and reproduction but will also generate several ground-breaking technical achievements in cell biology and live imaging, that should benefit the entire plant science community.