Expansin-controlled cell wall stiffness regulates root growth in Arabidopsis
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Summary: The reviewers felt that this is important work because in vivo characterization of expansins has lagged far behind their in vitro characterization. However, both reviewers also made important points about additional controls and statistical comparisons that are required to fully interpret and appreciate the results that are presented here. It seems that the role of expansins in the plant cell wall may be complex and nuanced. However, it is clear from the author's discussion of their results that significant further experimentation is required to bring new insight to the function of expansins in mediating plant root growth.
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Abstract
Expansins facilitate cell expansion via mediating pH-dependent cell wall (CW) loosening. However, the role of expansins in the control of biomechanical CW properties in the tissue and organ context remains elusive. We determined hormonal responsiveness and specificity of expression and localization of expansins predicted to be direct targets of cytokinin signalling. We found EXPA1 homogenously distributed throughout the CW of columella/ lateral root cap, while EXPA10 and EXPA14 localized predominantly at the three-cell boundaries of epidermis/cortex in various root zones. Cell type-specific localization of EXPA15 overlaps with higher CW stiffness measured via Brillouin light scattering microscopy. As indicated by both Brillouin frequency shift and AFM-measured Young’s modulus, EXPA1 overexpression upregulated CW stiffness, associated with shortening of the root apical meristem and root growth arrest. We propose that root growth in Arabidopsis requires delicate orchestration of biomechanical CW properties via tight regulation of various expansins’ localization to specific cell types and extracellular domains.
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Reviewer #2:
In this manuscript, the authors combine genetic/hormonal manipulation of expansin expression, localization studies, and mechanical measurements of root cell walls to study how this family of cell wall-loosening proteins influences root growth and development. This is an exciting topic, since expansins have a long history of in vitro characterization, but their characterization in living plants has lagged behind. The localization patterns of EXPA1, EXPA10, EXPA14, and EXPA15 are depicted using mCherry fusion proteins, and are shown to be distinct from one another. Despite the wide range of interesting approaches described here, I have some important concerns about the work as it stands, in terms of providing new insights into how expansins actually influence root growth.
Major Comments:
One major concern is the lack of …
Reviewer #2:
In this manuscript, the authors combine genetic/hormonal manipulation of expansin expression, localization studies, and mechanical measurements of root cell walls to study how this family of cell wall-loosening proteins influences root growth and development. This is an exciting topic, since expansins have a long history of in vitro characterization, but their characterization in living plants has lagged behind. The localization patterns of EXPA1, EXPA10, EXPA14, and EXPA15 are depicted using mCherry fusion proteins, and are shown to be distinct from one another. Despite the wide range of interesting approaches described here, I have some important concerns about the work as it stands, in terms of providing new insights into how expansins actually influence root growth.
Major Comments:
One major concern is the lack of appropriate controls, statistical appropriateness, and reporting (e.g., defining "n" clearly in all cases) in this work. All comparisons should include wild type and no-treatment controls; for example, in Figure 8, no AFM images are shown for wild type or EXPA1 overexpression cells.
Figure 1-S1: there is no change in pEXPA1::nls:3xGFP - why is there this discrepancy with the EXPA1 qPCR result? This is not explained.
Figure 3-S1: The finding of a lack of colocalization between EXPA10 and CFW staining is not convincing, due to a lack of a control showing positive colocalization and a lack of quantification of the degree of colocalization (e.g., Pearson correlation coefficient between red/blue pixels). The authors use these data as a lynchpin for part of their discussion, but this lack of colocalization could simply be an artifact of chromatic aberration, etc.
L256: This statement is not supported by the statistical comparisons shown in Figure 5B-C. In Figure 5B, why does the WT show higher MOC with Dex than without? In Figure 5B-C, you do not compare 8-4 + Dex with WT + Dex statistically, which is the salient comparison, and instead compare each genotype with vs. without Dex. In addition, the fact that the pRPS5A>GR>EXPA1:mCherry line does not show a significant difference in BLS signal with Dex addition (Figure 5-S1) argues against a clearly established relationship between expansin expression and BLS signal. The data in Figure 5D-E are more informative, but there is no wild type control for these experiments.
In Figure 8, the AFM color code scales do not seem to match the graphs, in that the color scales range from 0-2 MPa, whereas the graph Y axes range from 0 to 3 e6 MPa (unless that is supposed to be 0-3 MPa, or 0 to 3 e6 Pa!). No-Dex controls are missing from 8B.
In the Discussion, the authors use the words "unclear" and "elusive", and "remains to be identified" to sum up their work, and this to me is an indication of the state of this work overall. Although some of the data are intriguing, they are neither conclusive nor explanatory in revealing the mechanisms of expansin-mediated growth control in roots.
Finally, the manuscript needs to be revised for proper English grammar, syntax, and style.
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Reviewer #1:
Expansins are mysterious cell wall proteins because they lack known hydrolytic activity but are somehow correlated with acid-induced cell wall loosening/extension and cell expansion. Here the authors catalog the tissue expression of several native promoter driven expansin-FP fusions (EXPA1, 10, 14, 15) and find partially overlapping expression patterns and evidence that some expansins are restricted to particular cell wall regions (e.g. tricellular junctions (Figs 1-4). Using Brillouin light scattering (BLS) microscopy they find that, contrary to several previous reports for EXPA1, EXPA1 overexpression induces tissue stiffening that is relatively independent of extracellular pH (Fig 5, 7). They corroborate these data using AFM of different cell walls in a similar tissue (Fig 8). Thus, EXPA1 overexpression results in shorter …
Reviewer #1:
Expansins are mysterious cell wall proteins because they lack known hydrolytic activity but are somehow correlated with acid-induced cell wall loosening/extension and cell expansion. Here the authors catalog the tissue expression of several native promoter driven expansin-FP fusions (EXPA1, 10, 14, 15) and find partially overlapping expression patterns and evidence that some expansins are restricted to particular cell wall regions (e.g. tricellular junctions (Figs 1-4). Using Brillouin light scattering (BLS) microscopy they find that, contrary to several previous reports for EXPA1, EXPA1 overexpression induces tissue stiffening that is relatively independent of extracellular pH (Fig 5, 7). They corroborate these data using AFM of different cell walls in a similar tissue (Fig 8). Thus, EXPA1 overexpression results in shorter roots (Fig 9). While BLS seems like an interesting technique for studying cell walls, essential controls are missing making it difficult to interpret these results.
Major Comments:
Expansins have traditionally been identified with promoting cell wall extension by loosening the cell wall under acidic conditions. Recent reports have corroborated this: Ramakrishna et al., 2019 showed decreased lateral root initiation in mutants, implying EXPA1 plays a role in loosening, while Pacifici et al 2018 showed decreased cell elongation in expa1 mutants and increased cell elongation in EXPA overexpression lines, but only when grown on low pH (pH 4) media. All of these results are consistent with EXPAs playing a role in cell wall loosening. By contrast, the authors here find that EXPA1 overexpression causes cell wall stiffening and reduced root growth, that low pH (pH 4) media decreases this stiffening (Fig 5). Their discussion of these discrepancies is insufficient. For example, how do their levels of EXPA1 overexpression compare to Pacifici et al., 2018? How can they reconcile the results in these previous papers with their study?
Since the authors only really see changes in BLS of their EXPA1 line with over 10,000x overexpression (their inducible EXPA1-mCherry line with "only" >100x expression relative to wild type does not cause significant changes to cell wall "stiffness"), it is unclear how sensitive this technique is to cell wall changes. Controls are required to interpret these BLS experiments. For example, a known mutant or overexpression line with increased cell wall stiffness and another with decreased cell wall stiffness.
It will also be important to document whether the authors can replicate the lack of changes to cell wall stiffness in the expa1 mutant using AFM.
It would be helpful to see a detailed correlation analysis between the new technique (BLS) and an established cell wall analysis technique (AFM) across multiple data points (i.e. positive and negative controls for cell wall stiffness changes).
These AFM values are also presented on a scale that is almost 7x higher than previous data from the authors (e.g. Peaucelle 2014 JoVE). Please discuss.
The authors are comparing BLS data from the inner longitudinal cell wall versus AFM data from the outer longitudinal cell wall, which have very different properties. Please discuss.
EXPA1 gene overexpression is determined 7 days after Dex induction, but BLS experiments are conducted on plants that have been induced for a much shorter time (e.g. 3h). What is the expression of the EXPA1 gene over this timeframe of induction? Ideally, the authors would also use an EXPA1 antibody to monitor protein levels, since this is what is actually relevant.
It is difficult to see from the BLS shift maps provided (e.g. Fig 5A) where in the root the authors are imaging. Given that this is a relatively new technique to the cell wall field, it would be helpful to provide additional images to provide context to readers.
"Data not shown" (e.g. trans-zeatin treatments, line 149; EXPA1 protein levels, line 360) must be included as supplemental figures or the claims removed from the manuscript.
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Summary: The reviewers felt that this is important work because in vivo characterization of expansins has lagged far behind their in vitro characterization. However, both reviewers also made important points about additional controls and statistical comparisons that are required to fully interpret and appreciate the results that are presented here. It seems that the role of expansins in the plant cell wall may be complex and nuanced. However, it is clear from the author's discussion of their results that significant further experimentation is required to bring new insight to the function of expansins in mediating plant root growth.
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