Exploring the complexity of complexes in stomata development

Most land plants have small pores in their leaf surface, called stomata, through which they take up needed CO2 from the air for photosynthesis. The development of these vital structures follows a specific pattern and is regulated by a group of three very similar transcription factors (proteins which regulate the activity of genes in the DNA). Each of these transcription factors activates a different set of genes at a specific developmental stage. How they do this, however, is still unclear because due to their similarity they can be expected to bind the same DNA sequences. This is a common problem in biology because many transcription factors belong to larger families and have siblings with similar characteristics. The answer to it probably lies in the proteins that interact with the transcription factors to form protein complexes, which and can be decisive for their DNA binding and activity. Posttranslational modifications, which are changeable attachments to a protein, like phosphate molecules or tiny modifier-proteins, could also be responsible. Stomata are an excellent system to study this problem, because small changes in the function of the involved transcription factors lead to visible changes in stomatal development. This makes it easy to determine the general function of a protein in stomatal development. The aim of this project is therefore to compare the proteins that interact with the key transcription factors of stomatal development and to identify posttranslational modifications on them in order to understand what determines their functional difference. To do this, proteins that interact or come in close contact with the transcription factors will be isolated from young leaves, either by pulling out whole transcription factor complexes or by first marking proteins that come in close contact with the transcription factor to pull out those. These proteins will then be identified by mass spectrometry and probed for their interaction with all three transcription factors. Posttranslational modifications will be identified with the help of antibodies. The most interesting proteins and modifications will then be tested for their general importance for stomatal development by looking for stomata defects in mutant plants and for their effect on the transcription factors by testing the DNA binding, the activity, and the stability of the transcription factors. This will help us to better understand the molecular basis for stomatal development and can be very useful for other scientists investigating the function of mature stomata. Because stomatal development is quite different in dicots and monocots, a model plant from each of those two big groups will be used in this study - Arabidopsis thaliana for dicots and Brachypodium distachyon for monocots - to reveal new evolutionary differences in their regulation of stomatal development.

In this project, we identified new developmental regulators of stomata, which are specialized cells in plant leaves, and successfully adapted an exciting new method for identification of protein interactions or local protein assemblies for use in plants. Stomata are small pores in the leaf surface of most land plants, through which they take up carbon dioxide from the air for photosynthesis. The development of these vital structures follows a specific pattern and is regulated by a group of three very similar transcription factors (proteins which regulate the activity of genes in the DNA). Each of them activates a different set of genes at a specific developmental stage. How they can do this is a big question, because due to their similarity they are expected to bind the same DNA sequences, a theme that is quite common for large transcription factor families. One possibility is that other proteins interact with the transcription factors to form protein complexes and influence their DNA binding and activity. We therefore identified and compared proteins that bind to the three transcriptional master regulators of stomata development in two distinct plant species: the dicot Arabidopsis thaliana and the monocot Brachypodium distachyon. As expected, some interaction partners were shared between two or all three transcription factors, but others were unique. These proteins are other transcription factors, including such that could form dimers with different DNA binding preferences, and co-regulators that can affect the DNA structure to make short- and long-term changes to the activity of affected genes. Other identified proteins could influence the stability or activity of proteins in the transcription factor complexes. There is some similarity between proteins identified in Arabidopsis and Brachypodium, indicating that parts of the regulatory mechanisms are conserved, but there are also differences that fit to differences in the development and patterning of stomata on the leaves of these plant species. Further functional analysis of novel regulators of stomata development will improve our understanding of this process in the future. A major achievement of this project, that benefits plant research in general, was the adaptation of an efficient proximity labeling protocol in plants using an enzyme called TurboID which was originally developed for animal research. TurboID works by attaching a small molecular marker to close-by (proximal) proteins which can then be used for isolation of these proteins. With this method, we identified proteins that form complexes with one of the transcription factors regulating stomata development and proteins that are in the cellular nucleus of developing stomata. The use of this methods is, however, not restricted to these two applications, but will allow researchers to address many different questions. Adaptation of TurboID for use in plants in this project, therefore, lays the foundation for future proximity labeling experiments in other studies and will have a big impact on plant research in many different areas.