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.
