Pectin methylesterase activity is required for RALF1 peptide signalling output

Reviewer #1 (Public Review):

Summary:

Rößling et al., report in this study that the perception of RALF1 by the FER receptor is mediated by the association of RALF1 with deesterified pectin, contributing to the regulation of the cell wall matrix and plasma membrane dynamics. In addition, they report that this mode of action is independent from the previously reported cell wall sensing mechanism mediated by the FER-LRX complex.

This manuscript reproduces and aligns with the results from a recently published study (Liu et al., Cell) where they also report that RALF1 can interact with deesterified pectin, forming coacervates and promoting the recruitment of LLG-FER at the membrane.

Reviewer #2 (Public Review):

Summary:

The study by Rößling et al. addresses the link between the biochemical constitution of the cell wall, in particular the methylesterification state of pectin with signalling induced by the extracellular RALF peptide. The work suggests that only in the presence of demethylesterifies pectin, RALF is able to trigger activation of its receptor FERONIA (FER).

Remarkably, the application of RALF peptides leads to rather dramatic FER-dependent changes in wall integrity and plasma membrane invaginations not observed before. Interestingly, RALF can be out-titrated from the wall by short pectin fragments. In addition, the study provides further evidence for multiple FER-dependent pathways by showing the presence of LRX proteins is not required for the pectin/RALF mediated signalling.

Strengths:

This work provides fundamental insight into a complex emerging pathway, or perhaps several pathways, linking pectin sensing, pectin structure and RALF/FER signalling. The study provides convincing evidence that pectin methylesterase activity is required for RALF sensing, indicating that the physical interaction of RALFs with the cell wall is important for signalling. Beyond that, the study documents very clearly how profoundly RALF signalling can affect cell wall integrity and membrane topology.

Weaknesses:

The genetic material used by the authors to strengthen the connection of RALF signalling and PME activity might not be as suitable as an acute inhibition of PME activity.

The PMEI3ox line generated by Peaucelle et al., 2008 is alcohol-inducible. Was expression of the PMEI induced during the experiments? As ethanol inducible systems can be rather leaky, it would not be surprising if PME activity would be reduced even without induction, but maybe this would warrant testing whether PMEI3 is actually overexpressed and/or whether PME activity is decreased. On a similar note, the PMEI5ox plants do not appear to show the typical phenotype described for this line. I personally don't think these lines are necessary to support the study. Short-term interference with PME activity (such as with EGCG) might be more meaningful than life-long PMEI overexpression, in light of the numerous feedback pathways and their associated potential secondary effects. This might also explain why EGCG leads to an increase in pH, as one would expect from decreased PME activity, while PMEI expression (caveats from above apply) apparently does not (Fig 3A-D).

At least at first sight, the observation that OGs are able to titrate RALF from pectin binding seems at odds with the idea of cooperative binding with low affinity, leading to high avidity oligomers. Perhaps the can provide a speculative conceptual model of these interactions?

I could not find a description of the OG treatment/titration experiments, but I think it would be important to understand how these were performed with respect to OG concentration, timing of the application, etc.

Reviewer #3 (Public Review):

In this important work, the authors show compelling evidence that the Rapid Alkalinisation Factor1 (RALF1) peptide acts as an interlink between pectin methyl esterification status and FERONIA receptor-like kinase in mediating extracellular sensing. Moreover, the RALF1-mediated pectin perception is surprisingly independent of LRX-mediated extracellular sensing in roots. The authors also show that the peptide directly binds demethylated pectin and the positively charged amino acids are required for pectin binding as well as for its physiological activity.

Some present findings are surprising; previously, the FERONIA extracellular domain was shown to bind pectin directly, and the mode of operation in the pollen tube involves the LRX8-RALF4 complex, which seems not the case for RALF1 in the present study. Although some aspects remain controversial, this work is a very valuable addition to the ongoing debate about this elusive complex regulation and signaling.

The authors drafted the manuscript well, so I do not have a lot of criticism or suggestions. The experiments are well-designed, executed, and presented, and they solidly support the authors' claims.