Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In the present work, the authors characterised the temperature dependence of the splicing of the MsndhB gene. Although the work is mainly descriptive and preliminary, even in its stage it provides valuable information about the temperature acclimation processes. Therefore, the manuscript can be accepted after the following modifications:

Introduction:

• Give the abbreviation for PPR when it was mentioned first. (line 51)
• Explain CEF only once (see lines 66 and 75)
• The role of CEF in cold tolerance has been suggested long time ago by Jean-Marc Ducruet et al., so his works should also be cited.

Results:

• Figure and Table legends must be more self-explanatory. Among others: which organ was used?
• Figure 4 A and B: These results do not show exactly the same pattern. Figure 4B shows a gradual increase in the prematured form, whereas Figure 4A shows a sudden jump. In contrast, the matured gene product is intense after 3 hours, which is not observed in Figure 4B. What is the reason for these contradictory discrepancies? This needs to be explained in the text.

Discussion:

• Briefly discuss the physiological meaning of the changes in the Chlorophyll a/b.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript examines whether chloroplast MsndhB RNA splicing in alfalfa responds to abiotic stress and whether splicing efficiency is associated with photosynthetic physiology. Using ‘Xinjiang Daye’ alfalfa, the authors cloned and characterized MsndhB, compared conservation across species, and measured pre-mRNA, mature mRNA, splicing efficiency, gas exchange, fluorescence, chlorophyll, ATP, and MDA under heat, cold, salt, and drought treatments. The main claim is that heat suppresses MsndhB splicing, cold enhances it, and salt and drought mainly affect transcript abundance. The study contributes descriptive evidence on temperature-responsive chloroplast post-transcriptional regulation in forage legumes.

• Lines 14 to 22: The abstract states that the study assessed high-temperature, low-temperature, salt, and drought treatments. However, the physiological measurements reported in the same sentence are only described later for temperature stress. The abstract should clearly distinguish stress treatments used for splicing analysis from those used for photosynthetic and physiological analysis.
• Lines 23 to 31: The abstract reports correlations between splicing efficiency and physiological indicators. However, it does not state that these are associations rather than evidence of functional control.
• Lines 85 to 94: The study aim is clearly stated. However, the rationale for including salt and drought is weak because the preceding paragraph mainly builds a temperature-stress argument for ndhB splicing.
• Lines 78 to 83: The introduction cites temperature sensitivity of ndhB splicing in tobacco, rice, and Arabidopsis. However, it does not define the precise knowledge gap in alfalfa beyond species difference.
• Lines 96 to 105: Section 2.1 title is incomplete because it ends with “of the MsndhB,” which is grammatically unfinished.
• Lines 101 to 104: The results state that the intron has canonical 5′-GT and 3′-AC splice sites and that the protein contains a 492-aa NADH dehydrogenase domain. However, no accession number or deposited sequence identifier is provided for the cloned MsndhB sequence.
• Lines 131 to 141: The claim that secondary and tertiary structure predictions are “essentially consistent” is not sufficiently informative because no quantitative model-quality parameters, template identity, or confidence values are reported.
• Lines 145 to 146: Table 1 contains inconsistent gene symbols such as “Mtndhb” and “MpdnhB,” while most other entries use “ndhB.”
• Lines 166 to 174: Figure 4 legend contains a typographical error, “marure mRNA,” which should be corrected to “mature mRNA.”
• Lines 194 to 202: Figure 5 describes low-temperature results. However, the legend repeatedly states “under high-temperature stress.”
• Lines 217 to 225: Figure 6 presents salt and drought stress results. However, its legend again states “under high-temperature stress” for RT-PCR, qRT-PCR, and splicing efficiency panels.
• Lines 203 to 215: The text concludes that salt and drought did not significantly affect splicing efficiency. However, it does not specify whether stress effectiveness was validated by independent physiological or molecular stress markers.
• Lines 241 to 249: Figure 7 concerns developmental stages and tissues. However, its legend repeatedly describes “high-temperature stress.
• Lines 228 to 237: The comparison across roots, stems, leaves, flower buds, flowers, and pods is difficult to interpret because pods were collected at the pod-setting stage, whereas the other tissues were collected at early flowering. The tissue effect may therefore be confounded with developmental stage.
• Lines 251 to 263: The interpretation that increased Ci indicates non-stomatal limitation is plausible. However, it is stated without supporting biochemical or photochemical evidence beyond gas exchange and fluorescence.
• Lines 316 to 319: Figure 11 describes low-temperature data in the text immediately above. However, the legend states “under high-temperature stress.
• Lines 267 to 270 and 284 to 287: The figure legends report mean ± SE for Figures 8 and 9, whereas the statistical methods state mean ± SD. The manuscript must use one dispersion measure consistently across figures and methods.
• Lines 326 to 333: The text states that MDA accumulation shows a similar temporal pattern to MsndhB splicing efficiency under high temperature. However, splicing efficiency reaches a minimum while MDA increases.
• Lines 334 to 341: The correlation analysis is based on time-course data. However, the manuscript does not report the number of observations used in each Pearson correlation or whether time-dependent autocorrelation was considered.
• Lines 407 to 420: The discussion proposes that reduced MsndhB splicing may decrease mature transcripts and affect NDH complex-associated functions. However, the study did not directly assess NDH-B protein, NDH complex abundance, or NDH-dependent cyclic electron flow.
• Lines 477 to 487: The authors acknowledge that NDH-B protein abundance, NDH complex activity, and RNA editing were not examined, which is appropriate. These limitations should also temper the abstract and conclusion, where the physiological role of MsndhB splicing is presented more strongly than the evidence supports.
• Lines 500 to 508: The abiotic stress treatments are described. However, the manuscript does not state the number of plants per biological replicate or whether samples were pooled before RNA extraction.
• Lines 575 to 581: The formula for calculating splicing efficiency is not shown, although the text says it is calculated according to “the formula.” The exact equation is required because different definitions of splicing efficiency can produce different interpretations of S and U transcript abundance.
• Lines 585 to 603: The physiological methods cite kit or instrument use but provide limited procedural details for chlorophyll, ATP, and MDA assays. Sample mass, extraction volume, wavelength or calibration procedures should be stated to allow replication.
• Lines 610 to 616: The statistical analysis states one-way ANOVA with Tukey’s HSD. However, the design includes treatment type and time-course factors.
• Lines 618 to 627: The conclusion states that the findings provide a basis to explore temperature-responsive mechanisms, which is supported. However, the phrase “potential role” should be retained consistently because functional causality was not tested.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The author characterized the MsndhB gene and its RNA splicing behaviour in alfalfa under temperature, salt, and drought stress. The topic is relevant to chloroplast post-transcriptional regulation in a legume forage crop, the experimental design (multi-stress, developmental stage, and tissue comparisons) is reasonably comprehensive. However, the manuscript in its current form has significant weaknesses that must be addressed before publication. The response letter from author must be with line number as modified in the manuscript (Modification must be highlighted in main text) and what he has modified.

1. Author should provide the Protein sequences of all genes used in Figure 2.
2. Author did not provide the direct evidence at the protein/complex level (NDH-B abundance or NDH complex activity) links splicing efficiency to the proposed functional consequences. It should be acknowledged as a limitation but weakens several causal-sounding interpretive statements in the Discussion. Author should use the soft language around untested links between splicing efficiency and NDH complex function/protein abundance, and explicitly flag the flowering-stage “high transcription, low splicing” interpretation as a hypothesis.
3. The regulatory factor(s) responsible for MsndhB splicing are not identified; the study is purely descriptive/correlative at the molecular level.
4. Multiple figure captions (Figs. 5, 6, 7, 11) contain uncorrected copy-paste text from the Fig. 4/Fig. 9 template, misstating the treatment condition shown. Author should Correct all figure captions (Figs. 5, 6, 7, 11) to accurately reflect the treatment/condition shown, rather than reusing Fig. 4/Fig. 9 wording.
5. The reference list contains number of duplicated entries and several citation-content mismatches in the Discussion that require a full citation audit.
6. Table 1 is internally inconsistent with the main text regarding which species show non-canonical splice sites. Author should reconcile Table 1 with the main text (add the missing Pisum sativum entry; clarify the VkndhB 5’-site anomaly).
7. The splicing-efficiency calculation formula is referred to but never actually provided. Author should provide the splicing-efficiency calculation formula in Section 4.7.
8. Author should clarify that the 8 exonic nucleotide substitutions relative to the reference genome are synonymous or non-synonymous.
9. The whole manuscript needs carefully English editing and Correct English/typographical errors throughout notably the recurring “marure” → “mature” typo).

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

Manuscript is very well written. It provides new insights into the connection between RNA splicing and physiological responses in alfalfa under different abiotic stress with emphasis on temperature stress.

I only have a minor comment for authors:

In results Figure 13 Please mark with A and B what is high and what is low temperature stress in figure caption.

In M&M section 4.2. Please describe control conditions.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed most of the previous comments satisfactorily. However, the following points require clarification before acceptance.

The authors should clarify how the relative quantities of mature and unspliced transcripts obtained using different qRT-PCR primer pairs were compared to calculate splicing efficiency. The amplification efficiencies of both primer pairs should be provided.

In Figure 7E, pods are still assigned a statistical significance letter, although they were excluded from the tissue comparison. This inconsistency should be corrected.

The Pearson correlation analysis was based on six time-point means, which may not be statistically independent. These correlations should be interpreted cautiously as temporal associations.

Most protein structure models were generated using AlphaFold-derived templates. This limitation should be acknowledged when discussing the reliability of the predicted structures.

The MDA and ATP assay methods should include sufficient details on calibration and calculation procedures to ensure reproducibility.

The authors should confirm that the reported GenBank accession number (QB044729) is publicly accessible and corresponds to the cloned MsndhB sequence.

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The authors have improved the manuscript according to the provided comments and can be considered for publication

Author Response

Comments 1: The authors have improved the manuscript according to the provided comments and can be considered for publication.
Response 1: We thank the reviewer for the positive evaluation of our revised manuscript.