Periodic Reporting for period 1 - EmBd (Unravelling the molecular mechanisms of monocot embryogenesis)
Periodo di rendicontazione: 2019-09-01 al 2021-08-31
One key question in plant biology is how a single-celled zygote develops into a functional organism consisting of different tissues and organs. Knowledge on axis formation and meristem specification during embryogenesis in plants has tremendously advanced in the last two decades having adopted the dicot plant Arabidopsis thaliana as a model organism. The mature seeds of monocot plants display dramatic different structures than those of dicot plants. However, how these structures are patterned during embryogenesis is largely unknown. Thus, while much has been learnt from studying Arabidopsis embryogenesis, it is currently not clear if any concepts can be directly transposed to monocot embryogenesis. This underlines the urgency to study monocot embryogenesis. We took a monocot grass, Brachypodium distachyon as a model to systematically study monocot embryogenesis, especially for understanding early patterning events, such as formation of the primary axis and specification of the meristem. This will greatly increase our knowledge on tissue specification and organ formation in monocots and broaden our understanding of the biological diversity of plant early development. In addition, it will provide the basis for future investigation of crucial genetic interactions between individual seed compartments (seed coat, embryo, and endosperms).
1) Embryo development in Brachypodium.
In order to acquire the canonical growth patterns of the Brachypodium embryo, we first tried to observe successively staged (zygote until mature stage) embryos using differential interference contrast (DIC) microscopy in the Bd21 genotype; unfortunately, this did not yield the desired results. To image exact cell geometries, such as cell size, shape and volume, the transgenic plasma membrane reporter line pZmUbi:PM-mCherry could not be used for 3D imaging due to its weak reporter expression. It failed to outline cell walls clearly, which is a prerequisite for 3D segmentation via morphographX. Instead, we dissected seed tips, directly below which the embryo is located and used a described ClearSee method to clear tissue, followed by renaissance 2200 staining; this finally yielded high quality confocal images (Fig. 1). We performed 3D segmentation on successively staged embryos until the early globular stage and found that the first division taking place within the zygote is asymmetric (Fig. 2). More analysis need to be done to draw firm conclusions for the following cell divisions, such as observing whether they adhere to specific rules or take place completely randomly.
2) The expression of auxin related factors during Brachypodium embryogenesis
We studied the expression pattern of phytohormone auxin related markers in the Brachypodium embryo, specifically the markers DR5:RFP, PIN1a-Citrine, PINb-Citrine and SoPIN1-Citrine, lines that we obtained from O'Connor et al. (2014). DR5 was detected in the early leaf embryo (Fig. 3B-C), and could not be observed prior to this (Fig. 3A). During the earliest developmental stages, DR5 is expressed in the maternal tissue surrounding the embryo (Fig. 3A). Later it accumulates in the QC of the root, root cap, and vasculature of the scutellum (Fig. 3B-E). We initially detected PINa in the inner cells of globular stage embryos (Fig. 3F-G), after which it is expressed in the QC of the root, root cap, and vasculature (Fig. 3H-L). PIN1b could not be detected at the early globular stage (Fig. 3M) and was first detected to be expressed in the inner cells and a number of epidermal cells of the globular embryo (Fig. 3N). After these initial phases it shows a broader expression domain than PIN1a, which includes expression within the suspensor cells (Fig. 3O-R). SoPIN1 is initially expressed in the apical half of the embryo (Fig. 3S-T), while later it expands to the root and scutellum. Taken together, DR5 and PIN1 gene expression could be detected much later in Brachypodium embryos than their homologues could be in Arabidopsis embryos. This is consistent with a previous report that showed DR5 and PIN1 to be expressed much later in Maize embryos. One possible explanation is that the current version of DR5 used in this study is not as sensitive as DR5v2, which can be further tested.
3) Main results and exploitation and dissemination of these results
So far we have A) partially obtained the developmental atlas by segmenting the early stages of Brachypodium embryos using MorphoGraphX, B) set up a working transformation system in the lab as a basis for developing the genetic tools needed for studying monocot embryogenesis, C) analysed the expression of auxin-related reporter lines.
For exploitation: more work needs to be done to exploit the results from this action. For dissemination: we are currently working on a manuscript that will include all the results from this action so far and aiming for publication in a scientific journal next year.
In order to acquire the canonical growth patterns of the Brachypodium embryo, we first tried to observe successively staged (zygote until mature stage) embryos using differential interference contrast (DIC) microscopy in the Bd21 genotype; unfortunately, this did not yield the desired results. To image exact cell geometries, such as cell size, shape and volume, the transgenic plasma membrane reporter line pZmUbi:PM-mCherry could not be used for 3D imaging due to its weak reporter expression. It failed to outline cell walls clearly, which is a prerequisite for 3D segmentation via morphographX. Instead, we dissected seed tips, directly below which the embryo is located and used a described ClearSee method to clear tissue, followed by renaissance 2200 staining; this finally yielded high quality confocal images (Fig. 1). We performed 3D segmentation on successively staged embryos until the early globular stage and found that the first division taking place within the zygote is asymmetric (Fig. 2). More analysis need to be done to draw firm conclusions for the following cell divisions, such as observing whether they adhere to specific rules or take place completely randomly.
2) The expression of auxin related factors during Brachypodium embryogenesis
We studied the expression pattern of phytohormone auxin related markers in the Brachypodium embryo, specifically the markers DR5:RFP, PIN1a-Citrine, PINb-Citrine and SoPIN1-Citrine, lines that we obtained from O'Connor et al. (2014). DR5 was detected in the early leaf embryo (Fig. 3B-C), and could not be observed prior to this (Fig. 3A). During the earliest developmental stages, DR5 is expressed in the maternal tissue surrounding the embryo (Fig. 3A). Later it accumulates in the QC of the root, root cap, and vasculature of the scutellum (Fig. 3B-E). We initially detected PINa in the inner cells of globular stage embryos (Fig. 3F-G), after which it is expressed in the QC of the root, root cap, and vasculature (Fig. 3H-L). PIN1b could not be detected at the early globular stage (Fig. 3M) and was first detected to be expressed in the inner cells and a number of epidermal cells of the globular embryo (Fig. 3N). After these initial phases it shows a broader expression domain than PIN1a, which includes expression within the suspensor cells (Fig. 3O-R). SoPIN1 is initially expressed in the apical half of the embryo (Fig. 3S-T), while later it expands to the root and scutellum. Taken together, DR5 and PIN1 gene expression could be detected much later in Brachypodium embryos than their homologues could be in Arabidopsis embryos. This is consistent with a previous report that showed DR5 and PIN1 to be expressed much later in Maize embryos. One possible explanation is that the current version of DR5 used in this study is not as sensitive as DR5v2, which can be further tested.
3) Main results and exploitation and dissemination of these results
So far we have A) partially obtained the developmental atlas by segmenting the early stages of Brachypodium embryos using MorphoGraphX, B) set up a working transformation system in the lab as a basis for developing the genetic tools needed for studying monocot embryogenesis, C) analysed the expression of auxin-related reporter lines.
For exploitation: more work needs to be done to exploit the results from this action. For dissemination: we are currently working on a manuscript that will include all the results from this action so far and aiming for publication in a scientific journal next year.
From the current results, we know that the auxin signaling pathway likely plays a minor role in apical and basal axis formation during monocot embryogenesis. Since the project has been stopped prematurely, no novel results will be obtained. Based on the results thus far, we did see the potential of Brachypodium as a monocot model species and observed initial differences between dicot and monocot in embryo patterning, which would raise scientific attention to further investigate this direction.