Rebuilding core abscisic acid signaling pathways of Arabidopsis in yeast

Abstract The phytohormone abscisic acid (ABA) regulates plant responses to abiotic stress, such as drought and high osmotic conditions. The multitude of functionally redundant components involved in ABA signaling poses a major challenge for elucidating individual contributions to the response selectivity and sensitivity of the pathway. Here, we reconstructed single ABA signaling pathways in yeast for combinatorial analysis of ABA receptors and coreceptors, downstream‐acting SnRK2 protein kinases, and transcription factors. The analysis shows that some ABA receptors stimulate the pathway even in the absence of ABA and that SnRK2s are major determinants of ABA responsiveness by differing in the ligand‐dependent control. Five SnRK2s, including SnRK2.4 known to be active under osmotic stress in plants, activated ABA‐responsive transcription factors and were regulated by ABA receptor complexes in yeast. In the plant tissue, SnRK2.4 and ABA receptors competed for coreceptor interaction in an ABA‐dependent manner consistent with a tight integration of SnRK2.4 into the ABA signaling pathway. The study establishes the suitability of the yeast system for the dissection of core signaling cascades and opens up future avenues of research on ligand‐receptor regulation.

Thank you for submitting your manuscript for consideration by the EMBO Journal. I sincerely apologise for the unusual delay in the assessment of your work due to belated submission of referee reports. We have now received two reports on your manuscript, which are included below for your information.
As you will see from the comments, both reviewers appreciate the work and the quality of the data and recommend publication of the manuscript. Given these positive evaluations from two experts of the field, I would like to invite you to submit a revised version of your manuscript, in which you address the comments of reviewer #2. In general, a nice piece of work showing that yeast can be used to study the plant ABA signaling machinery. Grill's earlier discovery of the ABA receptor is perhaps one of the most important findings in plant basic research in the past two decades, up there with Estelle's discovery of the auxin receptor. The discovery that phosphatases are the direct target of the ABA receptor and that a kinase is also intimately involved are paradigms not seen in animal systems (yet), in terms of a protein phosphatase that is DIRECTLY regulated by a hormone receptor. Despite my great enthusiasm for Grill's earlier work and for the careful study described in this manuscript I am still left wondering whether the yeast system has really added a great deal of additional information not available with the protoplasts. However given the fact that yeast has not been used to reconstruct this machinery before and that without question it offers a great many new approaches to using synthetic biology for studying key aspects of the ABA dependent phosphatase and kinase, I highly recommend acceptance of this paper. This is not only excellent basic research but also, since ABA controls the movement of water out of a plant via stomata and also, so many other aspects of plant life (embryo desiccation and then germination), that this will undoubtedly also prove to be a very important aspect of translational efforts for improving plant water use efficiency.

Referee #2:
This is a very interesting manuscript by Ruschhhaupt and colleagues. Here, the authors analyzed the core abscisic acid (ABA) signaling factors of Arabidopsis thaliana in yeast to dissect ligand-receptor specificities in a functional and multiplexed approach.
This study is very compelling and elegant. The authors took advantage of the yeast system to rapidly test many different combinations of pathway components and extract quantitative information. As mentioned in the manuscript, yeast was previously used successfully as a system to test many components of the auxin signaling pathways (Pierre-Jerome et al., PNAS 2014). In the present manuscript, Ruschhhaupt and colleagues go even deeper in the analysis of the ABA signaling core. They even identify SnRK2.1, 2.4, 2.5 and 2.10 (part of the subgroup I of the Arabidopsis SnRK2 protein kinase family) as putative candidates for being part of the ABA signaling core in Arabidopsis thaliana. The present manuscript should trigger new in planta studies, for example on roles of other SnRK2s in direct ABA signal transduction.
Overall, I believe the presented approach and results will be widely used and of broad interest to the research community.
Minor comments: 1) Line 135: the authors mention Fig. 1C but there is no such figure in the present manuscript.
3) Fig. 4C: is there any particular reason why OST1 was not used in this LUC-activity assay? OST1 is used throughout the manuscript for obvious reasons but not here. It should be included here as well. 4) Line 189: "subgroup II" is misspelled.
1st Revision -authors' response 1st Jul 2019 Referee #1: In general, a nice piece of work showing that yeast can be used to study the plant ABA signaling machinery. Grill's earlier discovery of the ABA receptor is perhaps one of the most important findings in plant basic research in the past two decades, up there with Estelle's discovery of the auxin receptor. The discovery that phosphatases are the direct target of the ABA receptor and that a kinase is also intimately involved are paradigms not seen in animal systems (yet), in terms of a protein phosphatase that is DIRECTLY regulated by a hormone receptor. Despite my great enthusiasm for Grill's earlier work and for the careful study described in this manuscript I am still left wondering whether the yeast system has really added a great deal of additional information not available with the protoplasts. However, given the fact that yeast has not been used to reconstruct this machinery before and that without question it offers a great many new approaches to using synthetic biology for studying key aspects of the ABA dependent phosphatase and kinase, I highly recommend acceptance of this paper. This is not only excellent basic research but also, since ABA controls the movement of water out of a plant via stomata and also, so many other aspects of plant life (embryo desiccation and then germination), that this will undoubtedly also prove to be a very important aspect of translational efforts for improving plant water use efficiency.
Referee #2: This is a very interesting manuscript by Ruschhaupt and colleagues. Here, the authors analyzed the core abscisic acid (ABA) signaling factors of Arabidopsis thaliana in yeast to dissect ligand-receptor specificities in a functional and multiplexed approach.
This study is very compelling and elegant. The authors took advantage of the yeast system to rapidly test many different combinations of pathway components and extract quantitative information. As mentioned in the manuscript, yeast was previously used successfully as a system to test many components of the auxin signaling pathways (Pierre-Jerome et al., PNAS 2014). In the present manuscript, Ruschhhaupt and colleagues go even deeper in the analysis of the ABA signaling core. They even identify SnRK2.1, 2.4, 2.5 and 2.10 (part of the subgroup I of the Arabidopsis SnRK2 protein kinase family) as putative candidates for being part of the ABA signaling core in Arabidopsis thaliana. The present manuscript should trigger new in planta studies, for example on roles of other SnRK2s in direct ABA signal transduction. Overall, I believe the presented approach and results will be widely used and of broad interest to the research community. We very much appreciate the input of the editor and both reviewers, and their positive feedback. We addressed the points raised one-by-one as stated below. Minor comments: 1) Line 135: the authors mention Fig. 1C but there is no such figure in the present manuscript. Response: Sorry, meant is Fig. 1B. 2) Lines 148-149: RCAR1 does not activate signaling in yeast without exogenous ABA ( Fig. 2A). However, this observation is true in Arabidopsis protoplasts (Fig. 2B).
Response: There is an approximately twofold induction of ABA signaling by RCAR1 in yeast (p < 0.001; one-tailed t-test; see also now Table 2 of the appendix).
3) Fig. 4C: is there any particular reason why OST1 was not used in this LUC-activity assay? OST1 is used throughout the manuscript for obvious reasons but not here. It should be included here as well.
Response: The data for OST1 have been presented in Fig. 1A and was therefore omitted. We include now the data for better comparison in Fig. 4C. 4) Line 189: "subgroup II" is misspelled. Response: corrected.
2nd Editorial Decision 9th Jul 2019 Thank you for submitting the revised version of your manuscript. The main issues have now been addressed and I am happy to inform you that your manuscript has been accepted for publication. Congratulations on a nice study! ! ! ! ! " common tests, such as t--test (please specify whether paired vs. unpaired), simple χ2 tests, Wilcoxon and Mann--Whitney tests, can be unambiguously identified by name only, but more complex techniques should be described in the methods section; " are tests one--sided or two--sided? " are there adjustments for multiple comparisons? " exact statistical test results, e.g., P values = x but not P values < x; " definition of 'center values' as median or average; " definition of error bars as s.d. or s.e.m.
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Analysis of large sample size experiments in yeast revealed that normal distribution occurred with an average s.d. of about 30%. For ABF transactivation studies in yeast we choose a sample size of 6 in total to analyze an effect of 3xsigma with a 0.05 α--probability and a power (1--β) of 90%. For comparisions of single core--cascades we increase n to 12 in general, to reduce the detectabel effect size to 1.5 x sigma. These parameters were evaluated using the G*Power 3.1.9.4 software for an unpaired, two--sided t--test.
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