Dissecting the Role of Cell Wall Changes in Chilling Injury-Induced Gel Formation, Rubberiness, and Mealiness in Apricots

Round 1

Reviewer 1 Report

1. Space should be added between figures and unit in the text;

2. Line 195-196, is there any references about the criteria about the eat-soft and over-soft?

3. Line 230-235,method?

4. A general description of the figure 2,3,4, and 5 is necessary;

5. Ethylene plays important role during fruit softening and the authors have discussed in discussion section, what’s the variation of ethylene chilling injury?  

6. Line 374-378, method?

7. Is there any picture about the fruit after CI? It can help the reader to understand the phenotype of the CI.

Comments for author File: Comments.pdf

moderate editing of english language is required.

Author Response

Comments and Suggestions for Authors

1. Space should be added between figures and unit in the text;

Care was taken that a space was added between “Figure” and “unit” in the text (e.g. Figure 1A) to match the style of the Journal.

 

2. Line 195-196, is there any references about the criteria about the eat-soft and over-soft?

A definition of over-soft is referenced in Materials and Methods at line 118:

“… until fruit were over-soft at firmnesses of less than 10 N [11].”

 

3. Line 230-235, method?

The method has been given in the legend of each Figure that uses gel filtration, and in the method section lines 163-168.

 

4. A general description of the figure 2,3,4, and 5 is necessary

The authors have added a general title to each of those figures.

 

5. Ethylene plays important role during fruit softening and the authors have discussed in discussion section, what’s the variation of ethylene chilling injury?  

We have included a paragraph on variations of chilling injuries related to ethylene (lines 517-531) and a paragraph how ethylene reduces chilling injury in some fruit but increases them other fruit (lines 543-559) in the discussion.

 

6. Line 374-378, method?

This was part of the legend of Figure 5 – we have fixed this.

 

7. Is there any picture about the fruit after CI? It can help the reader to understand the phenotype of the CI.

We have included a photo of gel formation in supplementary material Figure S1. Since rubberiness and mealiness are textural changes, they are not possible to show in a photo.

 

 

 

Reviewer 2 Report

This is an important work on distinct pectin degradation modes in normally ripened and chilling-injury-treated fruits of apricots. This work also provides information about difference in pectin degradation during ripening process among stone fruit species.

 

Before recommending acceptance of this manuscript, I would ask the authors to consider my comments below.

 

Introduction

P2L53: An explanation of “20 N” should be required here.

 

Materials and methods

P3L132: “as described in [14]” → as described previously [14]

 

Results

Figure S1, 2 and P7L226-235, P9L265-274: There is no need for inclusion of supplemental figures and legends in the main manuscript.

 

P7L241-P8L247 and Figure 3: “After ripening subsequent to cold storage, cells in maximum chilling-injured fruit at eating firmness (5-15N) had developed more irregular shapes with some apparent collapse, and the extent of both intercellular spaces and cell-cell separation had increased” →I would ask the authors, if it possible, to quantify the areas of intercellular spaces and cell-cell separation because the difference is not so clear in the images (Figure 3 B and C).

 

P10L291-P8L293 and Figure 4: “The extent of cell wall separation and of intercellular spaces was much greater in the samples exhibiting chilling damage (Figure 4E, F) than in samples without (Figure 4C, D)” → As I asked previously, the author may quantify the areas of intercellular spaces to support their observation.

 

P11L338; “…and was most intense in fruit with all three CI symptoms (Figure 4E, F)” → …and the JIM7 staining of highly-esterified pectin was most intense in fruit with all three CI symptoms (Figure 4F)”

→ It is difficult to observe the difference in fluorescence intensity from JIM5 in the fruit with all three CI symptoms and other samples.   

 

P12L374-P13L378: The description may be the part of Figure 5 legend.

 

Discussion

 

P13L388: “… cell wall progressively weaker” → … cell wall progressively weaker (Figures 1A and 2A)

 

P13L389-390: “… with cell walls at harvest” → … with cell walls at harvest (Figures 3 A, B and S2A, B)

 

P13L393: “… that ripened without CI” → … that ripened without CI (Figure 5E, G)

 

P13L397-398: “…to the cell wall (NSP pectin in CWR), rendering…” → …to the cell wall (NSP pectin in CWR) (Figure 5 G), rendering…

 

P13L407: “…in this study” → …in this study (Figure 3F)

 

P13L411-412: “…at a similar firmness, but differences were evident for mean molecular weight of WSP” → …at a similar firmness (Figure S1D), but differences were evident for mean molecular weight of WSP (Figure S1A)

 

P13L423-424: “middle lamella-related CSP did not disappear from cell junctions, and degradation of this pectin was also not noticeable” → …middle lamella-related CSP did not disappear from cell junctions, and degradation of this pectin was also not noticeable (Figures 4 and 5)

 

P14L435-436: “…approximately 10N,” → …approximately 10N (Figure 2E),

 

P14L437: “…while ripening at 20℃,” → …while ripening at 20℃ (Figure 2A),

 

P14L446-450: “The lower UA content of the WSP extract, …at harvest instead of decreasing,…” → The lower UA content of the WSP extract (Figure 1A), the accompanying higher UA content in the insoluble cell wall residue (Figure 1 B), the absent increase in molecular weight of WSP characteristics for apricots ripening at 20℃ to similar firmness (Figure 2 B), as well as the UA content of NSP remaining similar to that of fruit at harvest instead of decreasing (Figure 1 C),…

 

P14L459-460: “…no increase in MW distribution of WSP, and no decrease in MW distribution of CSP, …” → …no increase in MW distribution of WSP (Figure 2 B), and no decrease in MW distribution of CSP (Figure 2 D), …

→ In fact, the MW distribution of CSP of CI-treated fruit decreased at 41N compared with at 58N (H) (Figure 2B), although the authors said “no decrease” here. The description should be revised.

 

P14L462: “…WSP of larger MW distribution…” → …WSP of larger MW distribution (Figure 5 B)…

 

P14L462-463: “Apricots that showed gel formation only,…CSP that looked more degraded than fruit with any other combination of CI symptoms…” → Which result (figure) indicates this notion??

 

P14L464-465: “fruit with mealiness and gel formation after 3 weeks at 0℃ and CSP of smaller MW distribution than fruit with same CI symptoms ripened after 5 weeks of cold-storage at 0℃” → → Which result (figure) indicates this notion??

Author Response

Comments and Suggestions for Authors

 

This is an important work on distinct pectin degradation modes in normally ripened and chilling-injury-treated fruits of apricots. This work also provides information about difference in pectin degradation during ripening process among stone fruit species.

 

Before recommending acceptance of this manuscript, I would ask the authors to consider my comments below.

 

Introduction

P2L53: An explanation of “20 N” should be required here.

We have changed the text to

“Cell wall changes during apricot softening often involve reduction in molecular weight (MW) of CDTA-soluble pectin (CSP) when fruit harvested just before eating-ripeness (20 N) were coldstored for 42 days [9], but whether these fruit exhibited CI symptoms was not investigated.” We believe this makes it clearer that 20 N is just ‘a bit underripe’. (Line 75)

Materials and methods

P3L132: “as described in [14]” → as described previously [14]

Done

Results

Figure S1, 2 and P7L226-235, P9L265-274: There is no need for inclusion of supplemental figures and legends in the main manuscript.

The authors will remove all supplemental figures and legends from the main manuscript before resubmission.

P7L241-P8L247 and Figure 3: “After ripening subsequent to cold storage, cells in maximum chilling-injured fruit at eating firmness (5-15N) had developed more irregular shapes with some apparent collapse, and the extent of both intercellular spaces and cell-cell separation had increased” →

I would ask the authors, if it possible, to quantify the areas of intercellular spaces and cell-cell separation because the difference is not so clear in the images (Figure 3 B and C).

We agree with the reviewer that the difference is not very visible in this Figure but much better in the supplemental Figure S2 (now Figure S3) where a lower magnification was used (20x instead of 40x). Hence, the old supplemental Figure S2 is now in the main body of the manuscript as Figure 3, and we have highlighted the areas of cell-cell separation in red. Compared with fruit without CI at similar firmness (panel B), the increase in intercellular space is now easy to observe (panel C). We left panel F unmarked as to not obscure the presence of pectin matter in the intercellular spaces (see also Figure 4K and 4L with pectin staining using pectin-specific antibodies). The original, unmarked panels remain as Figure S3.

We have also rewritten result section 3.2 and included more detail on cell separation and intercellular spaces (colouring marks additional sentences) (lines 276-298):

“In firm ‘CluthaGold’ fruit at harvest, mesocarp cells had irregular shapes, with distinctly stained and undulating cell walls, considerable cell-cell separation and relatively large intercellular spaces at tricellular junctions. There was also staining in the region between separated cell walls (Figure 3A). After softening to 5 N at 20 °C, staining of the cell walls with toluidine blue was less intense, indicating loss of cell wall material during softening. Whereas cell wall swelling was comparable to that at the harvest stage, the extent of cell-cell separation seemed reduced, perhaps due to increased osmotic pressure due to sugar accumulation within the cells (Figure 3B). After ripening subsequent to cold storage, cells in maximum chilling-injured fruit at eating firmness (5-15 N) had developed more irregular shapes with some apparent collapse, and the extent of both intercellular spaces and cell-cell separation had increased, though some cells did remain in close contact (Figure 3C). Cell wall staining appeared comparable to that of fruit ripened at 20 °C and there seemed to be no appreciable cell wall swelling (Figure 3A, C). Cells from tissue with gel symptoms had a similar appearance as cells at the harvest stage although they were eating soft (Figure 3A, D), whereas cells from tissue with gel and mealy CI symptoms were of the appearance of cells from tissue without CI with a slight increase in cell separation and an increase in intercellular space (Figure 3B, E). Mesocarp cells from firm, rubbery, gel fruit after 5 weeks cold storage showed strong and sharp cell wall staining. Notable is the pectin staining of intercellular spaces (Figure 3F).

In Figure 3, cell separation and intercellular spaces are marked in red, whereas Figure S2 shows the unmarked mesocarp cells of ‘CluthaGold’.”

 P10L291-P8L293 and Figure 4: “The extent of cell wall separation and of intercellular spaces was much greater in the samples exhibiting chilling damage (Figure 4E, F) than in samples without (Figure 4C, D)” → As I asked previously, the author may quantify the areas of intercellular spaces to support their observation.

P11L338; “…and was most intense in fruit with all three CI symptoms (Figure 4E, F)” → …and the JIM7 staining of highly-esterified pectin was most intense in fruit with all three CI symptoms (Figure 4F)” → It is difficult to observe the difference in fluorescence intensity from JIM5 in the fruit with all three CI symptoms and other samples.   

We have removed these sentences, as we are describing the extent of cell separation in the section before in this version.

P12L374-P13L378: The description may be the part of Figure 5 legend.

Yes, it is. We fixed this.

Discussion

P13L388: “… cell wall progressively weaker” → … cell wall progressively weaker (Figures 1A and 2A)

 P13L389-390: “… with cell walls at harvest” → … with cell walls at harvest (Figures 3 A, B and S2A, B)

 P13L393: “… that ripened without CI” → … that ripened without CI (Figure 5E, G)

 P13L397-398: “…to the cell wall (NSP pectin in CWR), rendering…” → …to the cell wall (NSP pectin in CWR) (Figure 5 G), rendering…

 P13L407: “…in this study” → …in this study (Figure 3F)

 P13L411-412: “…at a similar firmness, but differences were evident for mean molecular weight of WSP” → …at a similar firmness (Figure S1D), but differences were evident for mean molecular weight of WSP (Figure S1A)

 P13L423-424: “middle lamella-related CSP did not disappear from cell junctions, and degradation of this pectin was also not noticeable” → …middle lamella-related CSP did not disappear from cell junctions, and degradation of this pectin was also not noticeable (Figures 4 and 5)

 P14L435-436: “…approximately 10N,” → …approximately 10N (Figure 2E),

 P14L437: “…while ripening at 20℃,” → …while ripening at 20℃ (Figure 2A),

 P14L446-450: “The lower UA content of the WSP extract, …at harvest instead of decreasing,…” → The lower UA content of the WSP extract (Figure 1A), the accompanying higher UA content in the insoluble cell wall residue (Figure 1 B), the absent increase in molecular weight of WSP characteristics for apricots ripening at 20℃ to similar firmness (Figure 2 B), as well as the UA content of NSP remaining similar to that of fruit at harvest instead of decreasing (Figure 1 C),…

 P14L462: “…WSP of larger MW distribution…” → …WSP of larger MW distribution (Figure 5 B)…

These have been added.

P14L459-460: “…no increase in MW distribution of WSP, and no decrease in MW distribution of CSP, …” → …no increase in MW distribution of WSP (Figure 2 B), and no decrease in MW distribution of CSP (Figure 2 D), …

Added,

and

→ In fact, the MW distribution of CSP of CI-treated fruit decreased at 41N compared with at 58N (H) (Figure 2B), although the authors said “no decrease” here. The description should be revised.

We have revised the description accordingly (lines 509-512):

“ ‘Clutha Gold’ apricots that were mealy, rubbery and had gel, showed no increase in MW distribution of WSP (Figure 2B), and no decrease in MW distribution of CSP, except straight after removal from cold storage (Figure 2D and Figure S2D), compared with fruit that ripened normally without CI.”

The description was correct in the Result section (lines 248-254):

“The MW distribution of CSP, representing pectin largely derived from the middle lamellae, dropped considerably while fruit ripened to 38 and 41 N respectively, regardless of cold storage or ripening at 20 °C straight from harvest. In normally ripening fruit, the MW distribution continues to decrease (Figure 2C, S2B, C), whereas in coldstored fruit, the MW distribution of CSP is increasing again while CI symptoms like gel, mealiness and rubberiness further develop (Figure 2D, S2E,F).”

 P14L462-463: “Apricots that showed gel formation only,…CSP that looked more degraded than fruit with any other combination of CI symptoms…” → Which result (figure) indicates this notion??

“Figure 5D” added to Discussion (lines 514-516):

“Apricots that showed gel formation only, had WSP of larger MW distribution (Figure 5B) and CSP that looked more degraded than fruit with any other combination of CI symptoms (Figure 5D).”

 P14L464-465: “fruit with mealiness and gel formation after 3 weeks at 0℃ and CSP of smaller MW distribution than fruit with same CI symptoms ripened after 5 weeks of cold-storage at 0℃” → → Which result (figure) indicates this notion??

 We have removed this sentence.

Reviewer 3 Report

The authors investigated the properties of cell wall in apricot under CI stress. The topic is interesting. Although the alteration of cell wall in chill injured-fruit have been well elucidated, the study of changes of rubberiness and mealiness in apricot provide a new idea of the effect of CI on texture of fruit. However, some problems need to be addressed before further consideration.

1. As a climacteric fruit, ethylene production and respiratory rate are closely related to postharvest quality of apricot fruit. CI changes ethylene production and respiratory rate of fruit during storage, which should be measured in this experiment. Moreover, the relationship between ethylene production and texture also needs to be discussed.

2. Were the disease or healthy tissues taken for sampling ? Please clarify it.

3. Table 1 needs to be performed statistic analysis.

4. Please clarify the maturity of apricot used in the experiment, including firmness, TSS, color, etc.

5. The reason and mechanism of CI occurrence in fruit should be elucidated in the discussion.

6. The order of results should be consistent with material and methods section.

7. There are not enough references in the discussion, which is not up to standard in scientific research, such as line 391-399, line 417-427 (only one Ref. cited ), line 441-453 (only one Ref. cited) and line 455-465.

8. The significant analysis should be performed in the group and between the groups. 

English of the manuscript can be improved.

Author Response

Comments and Suggestions for Authors

The authors investigated the properties of cell wall in apricot under CI stress. The topic is interesting. Although the alteration of cell wall in chill injured-fruit have been well elucidated, the study of changes of rubberiness and mealiness in apricot provide a new idea of the effect of CI on texture of fruit. However, some problems need to be addressed before further consideration.

1. As a climacteric fruit, ethylene production and respiratory rate are closely related to postharvest quality of apricot fruit. CI changes ethylene production and respiratory rate of fruit during storage, which should be measured in this experiment. Moreover, the relationship between ethylene production and texture also needs to be discussed.

In this manuscript, the authors wanted to dissect the role of cell wall changes in chilling injury-induced gel formation, rubberiness and mealiness. We have not measured ethylene production at any stage of ripening, as the manuscript is not about the role of ethylene but finding differences between the various CI symptoms. However, we have discussed the role of ethylene in the Discussion in lines 517-559, and we also added discussion on the relationship between ethylene and texture (lines 517-531).

2. Were the disease or healthy tissues taken for sampling ? Please clarify it.

For sampling, the CI-affected tissues were taken. We have clarified this in the method section.

“For those fruit samples, immediately after phenotyping, the skin was removed, and sections of affected apricot flesh cut vertically from outer to inner cortex in the same location within the fruit to ensure tissue similarity.” Lines 173-175

3. Table 1 needs to be performed statistic analysis.

The authors excuse the oversight – we have added standard deviations to the averages in Table 1.

4. Please clarify the maturity of apricot used in the experiment, including firmness, TSS, color, etc.

We have added in the information about the fruit firmness and SSC at harvest in lines 115-116.

5. The reason and mechanism of CI occurrence in fruit should be elucidated in the discussion.

We have elucidated the reason and mechanism of CI occurrence in the abstract in lines 40-45, and throughout the Discussion for example in lines 441-448, 463-480 and 496-502.

6. The order of results should be consistent with material and methods section.

The order of Results and M&M sections is the best we were able to provide, as for some Result sections, more than one of the described methods has been used, notably sections 3.1 and 3.4.

7. There are not enough references in the discussion, which is not up to standard in scientific research, such as line 391-399, line 417-427 (only one Ref. cited ), line 441-453 (only one Ref. cited) and line 455-465.

Lines 391-399 – here we are discussing our own results; no references necessary here.

Lines 417-427 – also here we are discussing our own results, and the reference at the end of the section refers to nectarine and peach. We have clarified this (line 500).

Line 441-453 – also these lines deal with our own results; the study that publishes that cell wall enzymes did not recover in tomato ripening after cold storage is unique to our best knowledge.

Lines 455-465 – Here, we have mentioned two references that deal with the relatively narrow topic of having investigated mealiness and size distribution of peach and plum.

Throughout the Discussion, we have however added a further six references, and another in the M&M section.

8. The significant analysis should be performed in the group and between the groups. 

Statistical analyses were performed were appropriate.

 

 

Round 2

Reviewer 1 Report

Line 14, In should be unbold.

Line 228-229 should removed.

Line 263-264 is more appropriate to move to Figure legends.