expression changes during the suspensor to embryonic cell fate conversion. We expressed a stabilized mutant Aux/IAA12 (bdl) protein – a transcriptional inhibitor of auxin response (Hamann et al., 1999; Rademacher et al., 2012) - exclusively in suspensor cells using the GAL4/UAS two-component expression system to locally inhibit auxin response (Rademacher et al., 2012). For that purpose, we used an enhancer trap line (M0171) that expressed GAL4 specifically in suspensor cells. We next performed a genome-wide transcriptomic analysis on embryos harvested when the first aberrant cell divisions started to occur. Despite this narrow window in which the embryos were collected, an unexpectedly large number of genes were differentially expressed in M0171>>bdl embryos, suggesting that reprogramming is a complex transcriptional response. Because the pro-embryo and suspensor are physically connected, even in these isolated embryos, transcriptional changes will likely include both primary effects in suspensor cells and secondary effects in pro- embryo cells. Therefore, a large-scale expression analysis was performed using promoter-GFP reporters for nearly 70 differentially expressed genes. This helped to identify a smaller set of genes whose changes in expression are a likely result of local auxin response inhibition in suspensor cells. Here, we focused on a set of 4 bHLH genes. A rationale for choosing these bHLH genes for in-depth analysis was that other members of the bHLH transcription factor family were previously reported to be involved in auxin-dependent development (Chandler et al., 2009; De Rybel et al., 2013; Schlereth et al., 2010). We showed that all these 4 bHLHs are indeed regulated by auxin, in an ARF-dependent manner, although only one, namely bHLH49, appeared to be an immediate auxin target. Indeed, when analyzing genome-wide transcriptional changes in bHLH49 misexpression and bhlh49 mutant roots, we found that the other bHLH genes are among the misregulated genes. This suggests that their auxin regulation is mediated by bHLH49. Phenotypic analysis showed that bHLH49 is also biologically relevant for auxin-dependent suspensor to embryo transformation. Overexpression of bHLH49 resulted in abnormal divisions and even formation of embryo-like structures in the suspensor, resembling the suspensor-specific bdl misexpression. Thus, the auxin-repressed bHLH49 gene is an important mediator of auxin-dependent suppression of embryo identity in suspensor cells. An important, yet unanswered question is what cellular process is triggered by bHLH49 to promote embryo development in the suspensor? Our analysis of the bHLH49-dependent transcriptome does not pinpoint a key cellular process that can illuminate its ability to target proliferation of suspensor cells. Nonetheless, the overlap of differentially expressed genes in both M0171 and bHLH49 microarrays hints towards the need of defined set of genetic regulators required to induce the
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switch from suspensor to embryonic cell fate. An in-depth analysis of the genes targeted by bHLH49, ideally in embryos, will likely help to define a cellular target process. bHLH49 is normally more strongly expressed in embryo cells, and is repressed by auxin response in the suspensor. An important question is whether the same genes are targeted by bHLH49 in its normal expression domain and in the ectopic suspensor domain. Another interesting observation is that although bHLH49 overexpression can trigger formation in embryo-like structures in suspensor cells and despite the severe post-embryonic defects, twin seedlings were never observed. This observation can be associated with dedifferentiation of the suspensor cells (erasure of their initial transcriptional program) into cells that can only divide, but cannot be transformed completely into functional embryo structures (installation of embryonic program).
In the past decades, a range of genes has been identified that are able to trigger embryogenesis. As reviewed in Chapter 1, even though some potential convergence points between embryo regulators were suggested, a more rigorous and systematic analysis in a uniform system is needed to establish the relevance of such convergence points. In Chapter 3, we used the predictable suspensor-derived embryogenesis to systematically assess the effect of known “embryo inducers” on embryo initiation process. We demonstrated that, apart from bdl (Rademacher et al., 2012) and bHLH49 (Chapter 2), from the 15 previously reported “embryo inducers” only RKD1 was capable of generating suspensor-derived twin embryos, reflecting the specificity of suspensor to embryo transition. It is remarkable, how a single transcription factor is able to override the inhibition of the embryonic program in the suspensor cells and to generate viable mature twin plants. In Arabidopsis, there are 14 RWP-RK genes, divided in two subfamilies NIN-like and RKD (Schauser et al., 2005). The RKD subfamily is composed of 5 members and except RKD1 two other members (RKD2 and RKD4) were previously also reported to post-embryonically promote embryogenesis (Koszegi et al., 2011; Waki et al., 2011), but none of them could confer embryonic fate to suspensor cells. Yet, the homology between members of the family implies presence of a specific RKD1 domain that might be required for induction of embryo formation in suspensor cells. Further analysis of RKD1 function, possibly including domain swaps between RKD1 and its close homolog RKD2, should help to dissect the unique capacities of RKD1. There are two possible scenarios that could explain the differences in embryo-induction potential between the homologs. Firstly, since the RKD proteins act as transcription factors, the range of targets recognized by each protein might be different. Another interpretation could be that the difference lies, not in their intrinsic potential, but rather in the level to which they should be misexpressed in the suspensor. It is conceivable that RKD1 is