PGENETICS-D-25-01211

The transcription factor TaWRKY58 coordinates growth and drought sensitivity in wheat by repressing TaLRR and TaBCS1

PLOS Genetics

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PLOS Genetics

Anne Goriely

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PLOS Genetics

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At this stage, the following Authors/Authors require contributions: Yazhou Zhang, Xinyao Cheng, Xinyu Yu, Anyu Gu, Xufei Zhao, Mei Deng, Guoyue Cheng, Qiang Xu, Qiantao Jiang, and Yuming Wei. Please ensure that the full contributions of each author are acknowledged in the "Add/Edit/Remove Authors" section of our submission form.

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Reviewer's Responses to Questions

Comments to the Authors:

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Reviewer #2: This manuscript presents a well-structured and significant study. The authors convincingly identify TaWRKY58 as a key transcriptional repressor that fine-tunes the trade-off between plant height and drought response in wheat. The combination of genetic, molecular, and phenotypic analyses is a major strength. The proposed regulatory module is novel and has clear implications for wheat breeding. However, few comments listed in below:

1. While the direct repression of TaLRR and TaBCS1 by TaWRKY58 is solidly demonstrated, the functional link between these targets and the observed phenotypes could be more deeply explored. The manuscript states TaLRR is a signaling protein and TaBCS1 is an energy metabolism protein, but how exactly their repression by TaWRKY58 leads to altered GA levels (for TaLRR?) and soluble sugar accumulation (for TaBCS1?) remains somewhat speculative. Thus, it will be good if co-authors could add data or a more detailed discussion on: for example, Does TaLRR mutation affect GA biosynthesis/signaling genes or pathways? Does TaBCS1 mutation directly impact mitochondrial function (e.g., ATP levels, ROS) or the expression of sugar metabolism genes under drought? Measuring GA levels in the Δtahr mutant and soluble sugars/energy status in the Δtabcs1 mutant would significantly strengthen the causal chain from TF to target to metabolic phenotype.

2. The observation that TaWRKY58 is stress-induced but its loss-of-function does not confer strong stress susceptibility (Fig. 4) is intriguing and well-discussed. However, the proposed "priming" or "buffering" hypotheses, while reasonable, are not directly tested. It will be good if the co-authors could analyze the kinetics of target gene repression (TaLRR, TaBCS1) following stress induction of TaWRKY58. Does transient TaWRKY58 induction lead to sustained repression of its targets? This would provide molecular evidence for a priming mechanism.

3. The drought tolerance data for the Δtawrky58 mutant is based on soluble sugar accumulation and survival cell counts (S5 Fig). More comprehensive physiological data would bolster the claim of "enhanced drought tolerance." It will be good if co-authors could Include standard drought physiology metrics for the key genotypes (WT, Δtawrky58, overexpression line), such as: Water loss rate (relative water content over time), Stomatal conductance or photosynthetic parameters under progressive drought, or a clearer yield component assessment (e.g., spike fertility, grain number/weight) after drought-recovery cycles, especially given the focus on "yield stability."

4. The figure legends often state "n ≥ 3 biological replicates" or similar. For critical phenotypic measurements (e.g., plant height, soluble sugar content), the exact 'n' for each genotype should be explicitly stated in the legend or figure. The "Material and Methods" states two independent experimental replicates were conducted; this should be clarified in the context of biological vs. technical replication.

5. Fig 3H & 4J: Please specify which specific GA(s) were measured (e.g., GA1, GA4).

Fig 4: The legend for panels H, I, J references "figure 4H." This is confusing. Please correct the labels (likely should be "in plants from H" or similar).

Fig 6A: It would be helpful to show TaLRR and TaBCS1 expression in the overexpression line as well, to confirm the repressive effect.

Page 13, Line 200-201: "We detected 754,059 TaWRKY58-bound peaks" – This number seems extremely high. Please clarify if these are raw peaks from sequencing or after stringent filtering. A brief comment on what this high number might imply (e.g., low binding specificity, or a truly global regulator) would be useful.

Page 16, Line 283-284: "Under drought stress, the expression levels of TaLRR and TaBCS1 were significantly upregulated..." This seems counterintuitive if TaWRKY58 is induced and represses them. Please clarify or discuss this point—it might indicate a feedback loop or stress-induced override of repression.

6. The discussion is strong and thoughtful. The model (Fig. 8) is helpful but could be slightly enhanced. Consider adding the metabolic outputs (GA, soluble sugars) and the resulting phenotypes (height, drought tolerance) to the schematic to make the pathway from molecular mechanism to agronomic trait even clearer.

7. DAP-seq: The method states genomic DNA was from 'Fielder,' but the study uses 'Kronos' and 'Jing411' mutants. Please justify the use of a different cultivar for this genome-wide assay and discuss potential limitations.

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: No

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Dear Dr Zhang,

We are pleased to inform you that your manuscript entitled "The transcription factor TaWRKY58 coordinates growth and drought sensitivity in wheat by repressing TaLRR and TaBCS1" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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Comments from the reviewers (if applicable):

Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #2: The authors have provided a thorough, well-organized, and respectful response to the reviewers’ comments. They have addressed nearly all points with a combination of: new experimental data (e.g., GA measurements in Δt alrr and Δt abcs1 mutants), clarifications in figures and legends, revisions to the discussion to strengthen mechanistic interpretation and acknowledgment of limitations and future research directions. The response is scientifically sound and appropriate for a revision. Two minor comments issues need to clarify:

1. Fig. 8 revision: Updated to include metabolic outputs (GA, soluble sugars) and phenotypes – good improvement. However, Duplicate/redundant text (e.g., pages 73–74, 80–81) appears to be a formatting artifact from merging revisions. These do not affect scientific integrity but should be cleaned up in final proof.

2. Comment 5. The authors chose not to add O-TaWRKY58 expression data to Fig. 6A to avoid redundancy. This is acceptable for me, because the GA specification and legend corrections are appropriate. The decision not to add overexpression data to Fig. 6A is justified and does not weaken the conclusion.

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Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information., and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #2: Yes

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Reviewer #2: No

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