Saline–Alkali Tolerance Evaluation of Giant Reed (Arundo donax) Genotypes Under Saline–Alkali Stress at Seedling Stage

Comments 1: [There are so many abbreviations in abstract without mentioning the full form at first use. correct it]

Response 1: [We appreciate your valuable comment and agree with your suggestion. Consequently, we have revised all instances of abbreviated terms in the abstract. These revisions can be found in the revised manuscript on page 1, paragraph 2, specifically between lines 18 and 36.]

Comments 2: [Why these regions were selected. Why only one biotype from Rwanda. Was it a check or some other reasons to choose it? Please mention the regions in the abstract and present the location coordinates here.]

Response 2: [We appreciate your valuable comment and agree with your suggestion. And we have mentioned the regions in the abstract. Actuary, in the previous research, we had studied the salt tolerance evaluation of a total of 12 genotypes of the A.donax from different regions and countries at axillary bud germination stage. The list of Code number and location is as follows:

The cluster analysis results indicated that LZ_No.1, LZ_No.3, and LZ_No.7 were categorized as highly salt-tolerant genotypes, whereas LZ_No.8 and LZ_No.9 exhibited moderate salt tolerance. In contrast, LZ_No.5, LZ_No.10, LZ_No.11, and LZ_No.12 were identified as low salt-tolerant genotypes, while LZ_No.2, LZ_No.4, and LZ_No.6 were classified as salt-sensitive. Considering the observed variation in salt tolerance, morphological phenotypes, and geographical distribution of A.donax, five representative genotypes were selected from the aforementioned groups, along with a commonly genotype (A. donax var. versicolor), resulting in a total of six genotypes for further investigation.

Moreover, the location coordinates were presented and the revisions can be found in n the revised manuscript from page 5, specifically from line 165 (Table 1)].

Comments 3: [Overall the introduction is not written well. Sentences are long and lack a reasonable structure. Authors need to throughly improve the introduction section.]

Response 3: [Agree. We have improved the introduction section to response this point. These revisions can be found in the revised manuscript from page 3 to page 5, specifically from lines 60 to 67; 77 to 80; 100 to 111 and 139 to 153].

Comments 4: [“1% potassium permanganate solution or 40 mg·L^-1 sodium hypochlorite solution for 5 minutes” which one was used in present study?]

Response 4: [We choose 1% potassium permanganate solution for surface disinfection. Cas number and manufacturer can be found in the revised manuscript on page 5 and page 7, specifically in lines 196].

Comments 5: [Did you check EC of the soil before and after treatment? pH needed to be checked as well. At final stage each treatment received how much salt and what was the EC and pH? it is important to mention and cannot be ignored?]

Response 5: [We appreciate your valuable comment and agree with your suggestion. In fact, we previously measured the pH of the mixed saline-alkali solution prior to treatment, which was determined to be 9.25. Additionally, we analyzed the electrical conductivity (EC) at different concentrations, obtaining values of 66.6 mS·cm⁻¹ (75 mM), 73.0 mS·cm⁻¹ (150 mM), and 81.3 mS·cm⁻¹ (225 mM), respectively. Given that this study focused on evaluating the physiological responses of different A.donax genotypes to saline-alkali stress rather than monitoring changes in soil properties before and after treatment, I did not consider it necessary to measure soil EC before and after the experiment]

Comments 6: [“Furthermore, the R/S ratio was significantly affected by the interaction of salinity × genotype (Table 2)” this sentence is not a proper way to explain the results.]

Response 6: [We appreciate your valuable comment and agree with your suggestion. Rather than serving as an explanation of the results, this sentence aims to emphasize that the interaction between salinity and genotype had a significant impact on the root-shoot ratio, underscoring the intricate relationship between environmental stress and genetic tolerance.]

Comments 7: [All the parameters in Table 2 need full forms in footnote or in the table]

Response 7: [We sincerely appreciate your insightful comment and fully agree with your suggestion. We have finished the full forms of all the parameters in Table 2. These revisions can be found in the revised manuscript on page 9, specifically from lines 337 to 339]

Comments 8: [all figures are not readable. correct these]

Response 8: [We sincerely appreciate your insightful comment and fully agree with your suggestion. We have improved the figures quality to enhance clarity and readability. These revisions can be found in the revised manuscript from page 10 to page 14, specifically in line 342, line 359, line 380 and line 413.]

Comments 9: [Discussion need to be improved]

Response 9: [We sincerely appreciate your insightful comment and fully agree with your suggestion. We have refined the discussion and conclusions to enhance logical coherence, strengthen academic rigor, improve readability, and make the narrative more compelling. These revisions can be found in the revised manuscript from page 16 to page 19.]

Comments 1: [Abstract is good but need to add key findings of physiological parameters based on PCA.]

Response 1: [We sincerely appreciate your insightful comment and fully agree with your suggestion. We have added key findings of physiological parameters based on PCA in the abstract. These revisions can be found in the revised manuscript on page 1, specifically between lines 37 and 39.]

Comments 2: [Introduction: Well written but need to little bit precise (Line 55-Lin 103)]

Response 2: [We appreciate your valuable comment and agree with your suggestion. And we have enhanced the language to improve the reading experience from page 3 to page 4.]

Comments 3: [Materials and Methods: Table. 1. Table should include separate column for source of Seed (Department name and country separately mention). Also, all genotypes should be in one column having total 04 columns (S. No. Code Number, Source and Country Name)]

Response 3: [Agree. We have revised Table 1 to include the genotype name, code number, location, source, and country name. These modifications enhance clarity and completeness. The updated table can be found in the revised manuscript on page 5, specifically in line 165].

Comments 4: [Section 2.8 Mention all sig levels used for tests as mentioned in Table 2.]

Response 4: [We appreciate your valuable comment and agree with your suggestion. The revised content now provides a more comprehensive explanation, which can be found in the updated manuscript on page 8, specifically in line 305].

Comments 5: [Results: Fig No. 1-4 recheck the font size and the LSD/Post hoc test letters used should be prominent in the respective graphs. use proper legends.]

Response 5: [We appreciate your valuable comment and agree with your suggestion. We have improved the figures quality to enhance clarity and readability. These revisions can be found in the revised manuscript from page 10 to page 14, specifically in line 342, line 359, line 380 and line 413.]

Comments 6: [Section 04: Discussion seems prolong and if the journal policy has no restriction, then please try to reduce the text]

Response 6: [We appreciate your valuable comment and agree with your suggestion. We have streamlined the discussion section by reducing overly detailed explanations to improve clarity and conciseness. Additionally, we have refined the presentation of the discussion to strengthen the persuasiveness of our argument.]

Comments 1: [Please give the full name for abbreviated (LZ, SPAD, Fv/Fm, Pn etc.)]

Response 1: [We appreciate your valuable comment and agree with your suggestion. Consequently, we have corrected all instances of abbreviated terms in the abstract. These revisions can be found in the revised manuscript on page 1, paragraph 2, specifically between lines 18 and 36.]

Comments 2: [ Please replace “The” with “the”, line 24.]

Response 2: [Agree. We have replaced “The” with “the” in page 1, line 25.]

Comments 3: [Please state the concentrations tested as the results will be discussed in the following paragraph]

Response 3: [We agree with your suggestion and have incorporated the different concentrations of the mixture solution into the abstract to enhance clarity and completeness. This revision strengthens the study's precision and can be found in the updated manuscript on page 1, specifically in line 23].

Comments 4: [Please rephrase the abstract so that it is clear which genotype was affected by salinity and alkalinity. As it is written, it is not clear what the result is.]

Response 4: [Agree. We have highlighted that LZ_No.1 exhibits the highest resistance to saline-alkali stress, whereas LZ_No.6 was the most severely affected, classifying it as a salinity-sensitive genotype. These revisions can be found in the revised manuscript from page 1, specifically from lines 41 to 43].

Keywords:

Comments 5: [Please replace "Principal component analysis; Membership function comprehensive evaluation" with a word that reflects the study.]

Response 5: [We agree with your suggestion and have replaced "Principal Component Analysis; Membership Function Comprehensive Evaluation" with "Saline-Alkali Tolerance Evaluation". This revision strengthens the manuscript’s coherence and can be found on page 1, specifically in line 49 of the revised version].

Introduction

Comments 6: [The introduction section has been documented, however it should be noted that the inclusion of a paragraph discussing a different plant that has been the subject of research in the relevant literature would greatly enhance the introduction section.]

Response 6: [We sincerely appreciate your valuable feedback and fully agree with your suggestion. To enhance the manuscript's coherence, we have added sentences discussing promising solutions for improving plant resilience and mitigating the adverse effects of environmental stressors. These revisions contribute to a more comprehensive discussion and can be found in the updated manuscript on page 4, specifically from lines 106 to 115].

Comments 7: [Please highlight the novelty of this study compared to another work.]

Response 7: [We agree with your suggestion and have highlighted the novelty of this study. These revisions contribute to a more comprehensive discussion and can be found in the updated manuscript on page 4, specifically from lines 138to 142].

Materials and Methods

Comments 8: [I suggest including a subchapter listing all chemicals used and the manufacturer for each.]

Response 8: [We agree with your suggestion and have incorporated a table into the manuscript detailing the chemical names, CAS numbers, manufacturers, and countries of origin. These revisions enhance the completeness of the study and can be found in the updated manuscript on page 6, specifically in line 196].

Comments 9: [What was the basis for the code given for the samples?]

Response 9: [We appreciate your valuable comment and agree with your suggestion. Actuary, in the previous research, we had studied the salt tolerance evaluation of a total of 12 genotypes of the A.donax from different regions and countries at axillary bud germination stage. The list of Code number and location is as follows:

The code number of samples was assigned based on the order of discovery and the timeline of the study. To ensure a comprehensive analysis, five representative genotypes were selected from the identified groups, considering variations in salt tolerance, morphological phenotypes, and geographical distribution of A.donax. Additionally, a widely recognized genotype (A.donax var. versicolor) was included, bringing the total to six genotypes for further investigation.

Comments 10: [Please give the name of the manufacturer, city, country for each equipment used.]

Response 10: [We agree with your suggestion and have specified the manufacturer, city, and country for each piece of equipment used. These revisions can be found in the updated manuscript from pages 6 to 9, specifically in lines 202, 204, 211, 216-217, 222, 232, 239, 247-248 and 257.]

Comments 11: [The method used to measure soluble sugars, proline and malondialdehyde using the kit is not accurate. Please indicate the accuracy and precision of the methods.]

Response 11: [We appreciate your valuable feedback and agree with your suggestion. We have refined the procedures for determining MDA, Pro, and SS content and optimized the language for better readability. These revisions can be found in the updated manuscript on page 7, specifically from lines 230 to 267.]

Comments 12: [Please give the physico-chemical characteristics of the loamy soil used in the pot experiments.]

Response 12: [We appreciate your valuable feedback and fully agree with your suggestion. The physico-chemical properties of the loamy soil are as follows: pH = 5.687 ± 0.139, ammoniacal nitrogen content = 8.568 ± 0.617 μg·g⁻¹, and nitrate nitrogen content = 27.489 ± 0.895 μg·g⁻¹. If it is allowed, additional details on the physico-chemical characteristics of the loamy soil should be provided after February 10^th, 2025.]

Results and discussion

Comments 13: [Please make the figures clearer, it is difficult to interpret.]

Response 13: [We appreciate your valuable comment and agree with your suggestion. We have improved the figures quality to enhance clarity and readability. These revisions can be found in the revised manuscript from page 10 to page 14, specifically in line 342, line 359, line 380 and line 413.]

Comments 1: [First of all, you missed a description of the overall status of the seedlings after 7 days of treatment. Did any plant (in relation to a specific salt concentration) show salt/osmotic stress symptoms? Otherwise, do you think that (if so, why?) the differences in plant growth and photosynthetic parameters are sufficient for the proposed salt-stress resistance ranking?]

Response 1: [We appreciate your valuable feedback and fully agree with your suggestion. After 7 days of saline-alkali stress, there were no signs of stress-induced leaf chlorosis, wilting, or desiccation observed in any A.donax genotypes; however, a reduction in plant height was noted. These revisions can be found in the updated manuscript on page 9, specifically between lines 312 and 315.]

Comments 2: [Lines 129-130 “The saline-alkali treatment was applied when the plants had grown to the stage of three pairs of leaves.” I imagine that the different genotypes had different starting plantlets height at this point; did you consider this when you registered final plant height?]

Response 2: [We agree with your suggestion. Plants exhibiting uniform growth within each genotype were selected for the saline-alkali stress treatment, with corresponding control groups established for each genotype to ensure reliable comparisons. These revisions can be found in the updated manuscript on page 5, specifically between lines 176 and 177.]

Comments 3: [Lines 231-236 Root-shoot ratio “The root-shoot ratio of LZ_No.1 decreased first at low concentration of saline-alkali stress, but increased and reached the maximum value at 225 mM, which indicated that the strategy of continuously increasing underground biomass was adopted to cope with the gradually increasing saline and alkali stress concentration, while most of other genotypes reached the minimum value at 225 mM.” Actually I see (Figure 1b) different -even opposite- responses to salinity. Please discuss what the diverging results of LZ_No.1, LZ_No.11 and LZ_Var could be attributed to. Furthermore, this is a ratio…did salt concentration affected the total length of the roots in a statistically significant way?]

Response 3: [We appreciate your valuable comment and agree with your suggestion. The root-shoot ratio (R/S) is the proportion of a plant's biomass allocated to the roots compared to the shoots. An increase in this ratio often indicates that the plant is investing more resources into root growth relative to shoot growth. This increase can be indirectly due to an increase in total root length through physiological and ecological responses. The R/S of LZ_No.1 initially decreased under low concentrations of mixed saline-alkali stress but subsequently increased, reaching its peak at 225 mM. This pattern suggests that at lower stress levels, LZ_No.1 prioritized aboveground growth as a strategy to mitigate adverse environmental conditions, leading to a temporary reduction in the R/S ratio. However, when the stress concentration exceeded 150 mM, shoot growth was significantly inhibited, prompting the plant to allocate more resources to root system development to enhance nutrient and water absorption. This shift resulted in an increased R/S ratio at higher stress levels. The R/S ratio of LZ_No.11 followed a similar trend to that of LZ_No.1. but at lower concentrations, there was no significant difference between the treatment and control groups, indicating that LZ_No.11 maintained a relatively stable growth balance under mild saline-alkali stress. In contrast, LZ_Var. exhibited a peak R/S ratio at 150 mM but experienced a sharp decline at 225 mM. This suggests that at extremely high salinity levels, the stress exceeded the plant’s physiological tolerance, causing irreversible damage to the root system. As a result, root growth was more severely inhibited than shoot growth, leading to a significant decrease in the R/S ratio. These findings highlight the differential stress tolerance mechanisms among the tested genotypes. These revisions can be found in the revised manuscript from page 16 to 17, specifically from lines 496 to 506].

Comments 4: [Figure 2. How do you explain the extreme differences found among treated and untreated plants for net photosynthetic rate (Pn), transpiration rate (Tr) and (f) intercellular CO2 concentration (Ci), and the neutral effects for SPAD values and maximum photochemical efficiency of 256 PS Ⅱ（Fv/Fm)?]

Response 4: [We appreciate your valuable comment. The significant differences in P_n, T_r, and G_s between treated and untreated plants indicate that saline-alkali stress has a profound impact on the gas exchange processes in A. donax. Under stress conditions, stomatal closure restricts CO₂ uptake and reduces transpiration, leading to a decline in both P_n and T_r. The observed increase in C_i further suggests that limited gas exchange is the primary constraint on photosynthesis. In contrast, the minimal variation in SPAD values and F_v/F_m indicates that the photosynthetic apparatus remains functionally stable despite stress exposure. The sustained F_v/F_m values suggest that the primary photochemical reactions within PSII were not significantly impaired, while the unchanged SPAD values imply that chlorophyll degradation did not occur within the experimental timeframe. These findings suggest that while A. donax retains its photosynthetic pigment content and photochemical efficiency under saline-alkali stress, gas exchange processes are more susceptible to stress-induced alterations. These revisions can be found in the revised manuscript on page 18, specifically from lines 573 to 582].

Comments 5: [Lines 253-254  “Ci value of the six genotypes seedlings decreased to the lowest at 150mM, but increased at 225 mM (figure.2c to f). Why intercellular CO2 concentration increased at 225 mM?]

Response 5: [We appreciate your valuable comment. A decreasing trend in intercellular C_i was observed, reaching its lowest point at 150 mM, followed by an increase at 225 mM, while SPAD and F_v/F_m values remained stable. This pattern indicates a shift from stomatal limitation to non-stomatal limitation in response to increasing saline-alkali stress. At 150 mM, the reduction in C_i was primarily attributed to stomatal closure, which restricted CO₂ diffusion into the leaf. However, the stability of SPAD and F_v/F_m values suggests that the photosynthetic machinery remained functional, indicating that photosynthesis was not significantly impaired at this stage. However, at 225 mM, the subsequent increase in C_i implies that biochemical limitations on photosynthesis (such as enzyme inhibition, ATP synthesis disruption, or impaired CO₂ fixation efficiency) became the dominant constraint. Despite this, the unchanged SPAD and F_v/F_m values indicated that chlorophyll content and PSII function were still maintained, suggesting that the damage was primarily metabolic rather than structural. These findings suggest that A. donax can maintain photosynthetic efficiency under moderate stress by regulating stomatal behavior but faces increasing metabolic constraints at higher salinity levels, which may eventually lead to a decline in overall photosynthetic performance if the stress persists or intensifies. These revisions can be found in the revised manuscript on page 18, specifically from lines 571 to 576]

Comments 6: [Lines 282-283 “SS content of four genotypes 282 reached the maximum value at 150 mM but decreased under 225mM except LZ_No.12 283 and LZ_Var” actually this decrease is statistically significant only for LZNo3]

Response 6: [We sincerely appreciate your insightful comment and fully agree with your suggestion. To enhance clarity, we have refined the presentation of our results. Compared to the control, the SS content of most A. donax genotypes significantly increased under a mixed salinity stress concentration of 150 mM, except for LZ_Var. This suggests that soluble sugars play a crucial role in osmotic regulation and stress adaptation when saline-alkali stress levels exceed 150 mM. In contrast, the SS content of LZ_Var. showed no significant changes as stress concentration increased. This finding indicates that excessive abiotic stress may disrupt the accumulation of soluble sugars in LZ_Var., possibly due to impaired metabolic regulation. Additionally, the plant may be diverting energy toward other defense mechanisms, such as antioxidant enzyme activity or osmo-protectant synthesis, to mitigate the adverse effects of extreme stress conditions. These revisions can be found in the revised manuscript from page17 to 18, specifically from lines 531 to 540.]

Minor observations

Comments 7: [Please check the quality (resolution) of Figure 1, (and especially) Figure 2 to 4]

Response 7: [We appreciate your valuable comment and agree with your suggestion. We have improved the figures quality to enhance clarity and readability. These revisions can be found in the revised manuscript from page 10 to page 14, specifically in line 342, line 359, line 380 and line 413.]

Comments 8: [Please check all the merged words (e.g. in the Abstract : A.Donax, 225mM, 150mM-In the Abstract, please explain what REC, MDA, Pro and SS SOD, POD and CAT stand for.]

Response 8: [We appreciate your valuable comment and agree with your suggestion. Consequently, we have corrected all instances of abbreviated terms in the abstract. These revisions can be found in the revised manuscript on page 1, paragraph 2, specifically between lines 18 and 36.]

Comments 9: [Line 87 “Giant reed (Arundo Donax), a species belongs to the Gramineae family, as known as a …” probably you meant “Giant reed (Arundo Donax), a species belonging to the Gramineae family, is known as a”]

Response 9: [Agree. We have revised the sentence for improved readability. The updated version can be found on page 4, line 124 of the revised manuscript.]

Comments 10: [Lines 88-89 “which can be cultivated in marginal in degraded and marginal lands” please delete the first “marginal”]

Response 10: [Agree. We have deleted the first “marginal”. The updated version can be found on page 4, line 126 of the revised manuscript.]

Comments 11: [Please check the “指标” index]

Response 11: [Agree. We have deleted the word “指标”. The updated version can be found on page 15, from lines 459 (Table 4) of the revised manuscript.]

Comments 12: [Line 252 “…decreased to the lowest at 225mM. compared with the those of..” Please change into “..decreased to the lowest at 225 mM compared with those of..”]

Response 12: [Agree. We have revised the sentence for improved readability. The updated version can be found on page 11, from lines 367 to 370 of the revised manuscript.]

Comments 1: [Title: you do not need to mention 'physiological response' in the title. Good enough to say: Saline-Alkali Tolerance Evaluation of Giant Reed（Arundo Donax）Genotypes Under Saline-Alkali Stress at Seedling Stage]

Response 1: [We appreciate your valuable feedback and agree with your suggestion. We have deleted “physiological response” in the title for greater precision and smoothness. This revision can be found in the updated manuscript on page 1, specifically between lines 2 and 3.]

Comments 2: [The word "index" is a Latin word. Even it is accepted to use "indexes" as a plural; the correct Latin plural is "indices". You used two forms in many places, use only "indices" everywhere, for example L21 and L24.]

Response 2: [Thank you for your insightful comment. We have implemented the suggested revisions, updating all instances of 'indices' throughout the manuscript. These changes can be found in the revised version on page 1 (line 24 and 26), page 4 (line 148), page 8 (line 303), page 9 (line 317), page 14 (line 447), page 16 (line 445), and page 16 (line 488).]

Comments 3: [Abstract: should contain enough information about the tested genotypes and the tested salinity levels.]

Response 3: [Agree. We have incorporated the suggested revisions by updating the information on the tested genotypes and salinity levels in the abstract. These changes are reflected in the revised manuscript on page 1, specifically in lines 18 to 19.]

Comments 4: [Introduction: L108-L112 presented some kind of conclusion for what was done. Instead, these lines should summarize the objectives of the work.]

Response 4: [Thank you for your valuable comment. We have refined the statement of the study's purpose and significance to better emphasize its research impact. These revisions are reflected in the revised manuscript on pages 4 to 5, specifically in lines 155 to 157.]

Comments 5: [All figures are not clear and not readable.]

Response 5: [Agree. We acknowledge the lack of clarity in the original image, which may have affected readability. To address this, we have enhanced the quality of the figures for improved clarity and comprehension. These revisions can be found in the revised manuscript from page 10 to page 14, specifically in line 342, line 359, line 380 and line 413.]

Comments 6: [Fig. 1 title is not correct. The figure does not show the effect of the growth observed but the effect of salinity on different genotypes………………………]

Response 6: [Agree. We have refined the title of Fig.1.The revision can be found in the revised manuscript on page 10 specifically between lines 344 and 348.]

Comments 7: [Table 2: explain symbols of the parameters]

Response 7: [We have refined the information on all parameters to improve clarity and precision. These revisions are reflected in the revised manuscript on page 9, specifically between lines 337 and 339.]

Comments 8: [Fig. 2 not readable, title is not understood]

Response 8: [Agree. We have improved all the figures quality to enhance clarity and readability. And we also revised the title of Fig.2. These revisions can be found in the revised manuscript on page 11, specifically between lines 360 and 365]