Autocrine regulation of stomatal differentiation potential by EPF1 and ERECTA-LIKE1 ligand-receptor signaling

Essential revisions:

1) Perform expression analyses to show that MUTE is induced after estradiol application and that this induction correlates with the increase and the decrease in expression of ERL1 and EPF1 respectively, hours after induction (Figure 2G and H).

This is a valid point. We have performed qRT-PCR to verify the induction of estradiol-inducible MUTE overexpression (revised Figure 2C). The induction kinetics of MUTE precedes that of ERL1.

The same for the control treatment. Regarding Figure 3 and Figure 4; carry out expression analyses to show that EPF1 is induced after estradiol application.

The RT-PCR of the estradiol-inducible EPF1 has been published previously (Lee et al., 2012 Genes Dev). In response to the reviewers’ comment, we have further performed qRT-PCR analysis to verify the induction kinetics of estradiol-inducible EPF1 overexpression in the time frame relevant to our MUTE reporter analysis. The data are now provided as Figure 3—figure supplement 1C.

As in Figure 3D, they should also quantify the expression of pSCRM-GFP and SCRM-GFP fusion proteins in the nuclei and for the data presented in Figure 3—figure supplement 2 and Figure 4.

As requested, we have performed quantitative time-course image analyses of individual nuclei expressing SCRMpro::nucGFP, SCRMpro::GFP-SCRM, SPCHpro::nucGFP, and SPCHpro::SPCH-GFP upon induced EPF1 overexpression (iEPF1) within 24 hour time frame (see revised Figure 3E, F, and Figure 4C, D). As we described in our original manuscript, 24-hour post induction of iEPF1 is sufficient to observe the complete disappearance of MUTE-GFP protein accumulation. As shown in the revised Figure 3E, F and Figure 4C, D, these reporter signals did not undergo obvious reduction due to iEPF1. In summary, the results from our large-scale image quantification fully support our original conclusion.

It is very difficult to produce large quantities of highly pure, properly-refolded bioactive EPF1 peptides. The disappearance of MUTE-GFP protein and consequential developmental arrests of meristemoids by induced EPF1 overexpression and EPF1 peptide treatments are highly reproducible. For this reason, further time-course quantification analyses of Figure 3—figure supplement 2 would add little insight to our findings.

2) Provide information as to how the marker lines were generated (transformation or crossing), plant generation used, expression levels of the respective tagged proteins and background used (wild type, mutant). It is not always clear what was the genetic background used to express the tagged proteins, for example in Figure 2A, are ERL1-YFP and MUTE-tagRFP expressed in the Col-0 or erl1 background? Similarly for Figure 2E.

ERL1pro::ERL1-YFP and MUTEpro::MUTE-tagRFP are expressed in erl1 and Col, respectively then subjected to genetic crosses. For all marker lines used for this study, we have provided detailed information about their genetic background in the revised Methods section and figure legend as appropriate.

3) Fluorescently-tagged receptors are not always functional (sometimes they are only partially functional, for example BAK1, FLS2); therefore is important that the authors quantify the phenotypes of the rescued er erl1 erl2 triple mutant (Figure 5), in order to be more convincing that the ERL1-GFP fusion is functional (similar experiments were done for pMUTE::ERL1-YFP introduced in the er erl1 erl2 triple mutant).

This is a valid criticism. In the original submission, we provided the confocal image of the cotyledon epidermis showing that ERL1pro::ERL1-YFP rescues the stomatal clustering phenotype of er erl1 erl2 triple mutant (Figure 1—figure supplement 1). As requested, we have performed quantitative analysis of stomatal index and clustering distribution, now provided as Figure 1—figure supplement 1B, C.

Also, the expression levels should be close to the endogenous, as even when using an endogenous promoter, overexpression of the transgene is frequently observed.

As requested, we have analyzed the expression level of ERL1pro::ERL1-YFP in the erl1 knockout background (revised Figure 1—figure supplement 1D). We detected a slight (~1.25 fold) but not significant elevation of the transgene expression compared to the wild-type ERL1 (p=0.074, Student's t-test). Since ERL1pro::ERL1-YFP rescues the stomatal clustering phenotype of er erl1 erl2 to the same extent as the endogenous ERL1 gene (p= 0.49, Tukey's HSD test. See Figure 1—figure supplement 1B), the transgene does not confer overexpression or ectopic effects

Is the MUTE-GFP fusion functional?

Yes. Complementation of mute by MUTEpro::MUTE-GFP was published previously (Pillitteri et al., 2007 Nature 445: 501-).

4) TagRFP is a slowly maturing protein (100 min, in contrast to 10 min for GFP) and this might affect the co-localization dynamics shown in Figure 2A and B, Figure 5. Provide data or discuss this possibility.

We have performed a careful analysis of MUTEpro::MUTE-GFP and MUTEpro::MUTEtagRFP expression during stomatal development. As shown in the revised Figure 2—figure supplement 1A, their signal accumulation patterns are essentially identical. In addition, during the revision process we did genetic crosses, generated a double transgenic F1 seedlings carrying both MUTEpro::MUTE-GFP and MUTEpro::MUTEtagRFP, and analyzed their signals. Again, both GFP and RFP signals are co-detected during meristemoid-to-GMC transition (revised Figure 2—figure supplement 1B).

Since we are analyzing their accumulation patterns during the merstemoid-to-GMC transition, which takes an average of 19 hours (see Figure 5), we believe that the ~90 minute differences in the maturation time for GFP vs. tagRFP would not affect the co-localization dynamics or interpretation of our results within the time scale of our analysis.

5) It is not clear why MUTE-GFP in the scrm-D background was used for the ChIP assays and not MUTE-GFP in wild type. Is SCRM required for MUTE binding? Explain or discuss.

As referenced in our original submission, the effective use of the scrm-D background for ChIP experiments was published previously (Horst et al., 2015 PLOS Genetics). The scrm-D mutation allows the entire protoderm to undergo stomatal cell-lineage transitions (Kanaoka et al., 2008 Plant Cell). Because nearly all epidermal cells follow the meristemoid-to-GMC transition, one could substantially increase the signals for MUTE-GFP ChIP assays. In the revised manuscript, we have explained rationales behind using the scrm-D background (subsection “ERL1 is a direct MUTE target”, second paragraph).

We have in addition performed ChIP experiments using Arabidopsis seedlings carrying functional MUTEpro::MUTE-GFP in mute null mutant background. This line was published previously, and the seedlings are phenotypically wild type (Pillitteri et al., 2007 Nature). We were able to detect the binding of MUTE-GFP to the ERL1 promoter region with lesser fold enrichment, owning to fewer cells undergoing meristemoid-to-GMC transition (Revised Figure 2—figure supplement 2C). The results further emphasize that MUTE binds to the ERL1 promoter during normal stomatal development (and therefore it is not the artifact of the scrm-D mutation).