Dissecting Rice Pearl Character, an Important Added Value in High-Quality Temperate Mediterranean Japonica Cultivars
Round 1
Reviewer 1 Report (New Reviewer)
The article presents novel results regarding pearled rice. Some minor revisions are suggested in order to improve the presentation of the research work:
Reference 22: add name of the journal.
Lines 156-157: Explain briefly the method with adaptations, so that other researchers could reproduce it.
Figure 3: it would result useful to add white and/or black arrows to show the different zones described in the results section (amyloplasts, starch granules, protein bodies). Add zoom used for the pictures. Is it the same for all the images?
Discussion: add some discussion regarding the amylose content, beyond its relationship with crystallinity. What is expected for each type of grain? Relationship with characteristics of the rice (hardness, stickiness)?
Lines 465-466: Review sentence.
Author Response
Response to Reviewer 1 Comments
Point 1: Reference 22: add name of the journal.
Response 1: Reference 22 has been corrected by adding the name of the journal
Point 2: Lines 156-157: Explain briefly the method used for protein extraction, so that other researchers could reproduce it.
Response 2: a Brief description of the protein extraction protocol was included:
“In a 50 ml tube, 500 mg of sample flour were mixed with 140 mL 40 mM phenylmethylsulfonyl fluoride (PMSF) and 10 ml of extraction buffer (0.7 M sucrose, 0.5 M Tris-base, 50 mM EDTA (pH 8.0), 0.1 M KCl (w/v), 5 mM HCl (v/v) and 0.328 M b-mercaptoethanol in miliQ water). The mixture was centrifuged (25,000 G 4ºC for 20 minutes) and the supernatant transferred to another fresh tube. Three times the volume of cold precipitation buffer (0.1 M NH4OAc and methanol, 10 mM b-mercaptoethanol and methanol) was added to the recovered phenol phase. After mixing by inversion, it was left incubating overnight at -20 ºC and the pellet was recovered and washed twice with 1.8 ml precipitation buffer by centrifugation (7,200 G 4ºC for 5 minutes). Then, the pellet was dried in an Eppendorf 5301 Concentrator Speed Vac system (Eppendorf, Hamburg, Germany) for 3 minutes and resuspended in 1.5 ml of lysis buffer (9.5M urea, 40 mM tris-base and 15 mM DTT in distilled water.”
Point 3: it would result useful to add white and/or black arrows to show the different zones described in the results section (amyloplasts, starch granules, protein bodies). Add zoom used for the pictures. Is it the same for all the images?
Response 3: Arrows indicating amyloplasts, free starch granules and protein bodies have been included in Figure 3. The zoom is already indicated for all images (2000 x) being the same for all of the pictures.
Point 4: Discussion: add some discussion regarding the amylose content, beyond its relationship with crystallinity. What is expected for each type of grain? Relationship with characteristics of the rice (hardness, stickiness)?
Response 4: Some discussion has been included regarding the amylose content as requested:
“The amylose/amylopectin ratio influences the physicochemical properties of a certain starch, affecting both gelatinization and retrogradation of starch from various botanical sources [1-3]. During gelatinization, starch granules swell and form gel particles. In general, starch granules are rich in amylopectin, the layers being interlaced with strands of open amylose chains. Upon swelling linear molecules of amylose diffuse out of the swollen granules making up the continuous phase (network) outside the granules [4]. It is well known that sticky starches usually swell to a greater extent than their non-sticky counterparts [5]. Amylose has been proposed to act as a restraint to swelling [4. The averaged amylose percentages varies for each cultivar [26], being also stated in our studied varieties. Montsianell is a low amylose variety specially indicated for Paella recipes (low stickiness) while Carnaroli has the highest amylose content, resulting ideal to attain the expected creaminess of the Risotto recipes.”
Point 5: Lines 465-466: Review sentence. The authors compared physicochemical of the so called “pearled rice” grains with crystalline rice.
Response 5: Sentence reviewed.
Author Response File: 
Author Response.docx
Reviewer 2 Report (New Reviewer)
This is an interesting manuscript investigating the characteristics of chalky (pearly) grain in different rice cultivars. Grain appearance in rice is important for processing, trading, and consumption; thus, this study is important at least in the local area. There are some recommendations, as follows:
1. The definition of white core, white belly, and pearly grain is not well clarified; in order to improve the readability, it is better to provide a more detailed explanation or charts.
2. The grains from 5 cultivars were used in this study; however, the background of the cultivation is not provided, as both environmental factors and agronomic management, such as fertilization, will affect grain chalkiness; this is the most important background information, which may decide the outcome of the entire study.
3. In the caption of Figure 1, it is better to clarify that the grains illustrated are milled grains.
Author Response
Response to Reviewer 2 Comments
Point 1: The definition of white core, white belly, and pearly grain is not well clarified; in order to improve the readability, it is better to provide a more detailed explanation or charts.
Response 1: The definition of white core, white belly and pealed grains has been better clarified within the abstract and the introduction to improve readability, although no chart has been thought to be included.
Point 2: The grains from 5 cultivars were used in this study; however, the background of the cultivation is not provided, as both environmental factors and agronomic management, such as fertilization, will affect grain chalkiness; this is the most important background information, which may decide the outcome of the entire study.
Response 2: The samples have been provided by farmers associations. Each variety samples come from hugh storage silos, being the mix of milled grains coming from hundreds of hectars cultivated with the same cultivar but form different farmers. Theese farmers are professional rice producers which apply the common agronomic practicies for each cultivar in their prodcution regions. Chalky grains are the result of the “sorting machines” that processed tons and tons of grains to reject unperfect grains.
Environmental factors don’t modify the white-core or white-belly characters, only the ammount of rejected stress-induced chalky grains. Thus, restrictive fertilizations may increase the rejection of chalky grains by the sorting machine after milling, although it might not affect the averaged physicochemical properties of chalky grains per se.
Point 3: In the caption of Figure 1, it is better to clarify that the grains illustrated are milled grains.
Response 2: Solved: “Figure 1. Milled rice grains photographs…”
Point 4: In the introduction the first paragraph should be summarized.
Response 4. We understant that we can’t further summarize the first paragraph, since it is key for clarifying the pealed white core and white belly definition as requested in Point 1. This clarification starts in paragraph one giving the main keys to further clarify those concepts in paragraph two.
Author Response File: Author Response.docx
This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.
Round 1
Reviewer 1 Report
The authors compared physicochemical of the so called “pearled rice” grains with crystalline rice. In my opinion, there is no such a group of “pearled” rice. The term “pearled” the authors used in the manuscript is chalky per se, whereas the term “chalky” is “floury” or “opaque” that has been wildly accepted and used in the community. Five cultivars were used for study, three of which are pearled and the rest two are crystalline. My criticism is that the sample size of each type was too small to get any convincible conclusions for characterization of the two types of rice.
The study is superficial, and does not deserve publishing at all.
Author Response
Response to Reviewer 1 Comments
Point 1: The authors compared physicochemical of the so called “pearled rice” grains with crystalline rice.
Response 1: By one hand, we compared chalky grains with the pearled fraction and the crystalline fraction of pearled (white-core and white-belly) grains from pearled varieties.
By the other hand, we compared chalky grains and crystalline grains from crystalline varieties. We could not compare pearled grains (white core grains) from crystalline varieties since they simply don’t exist, at least within the European cultivars.
Point 2: In my opinion, there is no such a group of “pearled” rice. The term “pearled” the authors used in the manuscript is chalky per se, whereas the term “chalky” is “floury” or “opaque” that has been wildly accepted and used in the community.
Response 2: In Europe we call pearled rice verities those having almost all grains with white core independently of the temperatures and dry winds suffered during grain filling. Contrary, chalkiness is derived from high temperatures and dry winds during the maturation of susceptible varieties and only present in few grains. All the pearled and crystalline varieties present few chalky grains, but crystalline varieties never show white core (pearled) grains. We specified now that Montsianell and Bomba are white-core cultivars whlie Carnaroli is a white-belly cultivar, using the term “stress-induced chalkiness” for those fluory grains.
Point 3: My criticism is that the sample size of each type was too small to get any convincible conclusions for characterization of the two types of rice.
Response 3: Having included more varieties wouldn’t statistically increase the samples sizes. We have included three different pearled rice grains: a medium-grain white-core variety (Montsianell), a short-grain white.core cultivar (Bomba) and a white-belly long A grain (Carnaroli). No comercial pearled long B grains exist since they would break during milling. Thus, they are a selection of independly breeded varieties, all other pearled varieties in Europe derive from theese three ones or are closely related genetically with those three ones.
Reviewer 2 Report
The abstract can be improved. Can be rewritten and focused on the main objective of the work
in the introduction the first paragraph should be summarized.
explain more clearly the confusion between pearled rice and rice chalkiness
in the results section 3.1- why cultivars Guadiamar and PL12 doesn’t have the pearl area percentage
min the section 3.2 make the figures a little bit bigger to show differences
the Discussion section the amylose content need to be clarify for more understanding why significant differences.
please summarize the conclusion
Author Response
Response to Reviewer 2 Comments
Point 1: The abstract can be improved. Can be rewritten and focused on the main objective of the work.
Response 1: The abstract has been deeply improved to focuss on the main objective of the work.
Point 2: in the introduction the first paragraph should be summarized.
Response 2: The first introduction paragraph has been reduced.
Point 3: explain more clearly the confusion between pearled rice and rice chalkiness
Response 3: It has been better explained the differences between white-core and white-belly pearled rice and stress-induced chalkiness.
Point 4: in the results section 3.1- why cultivars Guadiamar and PL12 doesn’t have the pearl area percentage
Response 4: Because they are christalline varieties, thus, no pearled area percentage can be provided. Christalline varieties only produce christalline grains with a tiny small fraction of chalky grains, never pearled ones.
Point 5: in the section 3.2 make the figures a little bit bigger to show differences
Response 5: Figure 2 size has been increased.
Point 6: the Discussion section the amylose content need to be clarify for more understanding why significant differences.
Response 6: The amylose content results have been better discussed, highlighting how different are white-core and white-belly pearled fractions with stress-induced chalky grains in terms of amilose content, which is the main difference between pearled and chalky grains together with the protein contents.
Point 7: please summarize the conclusion
Response 7: The conclusion has been summarized.
Reviewer 3 Report
*Third paragraph from down, add some references to the confused concept of rice pearl and rice chalkiness
* Have you used "Olesa" cultivar in this study ? Why is it present in fig1?
*At what height the rice grain images were captured and what imaging analysis software have you used?
*Title 2.7(Write it all)
*Results: 3.1 Discuss your results quantitatively.
Same comment for 3.5
*Indeed, Lin et al., ......... Check out this sentence
*Your results (X-Ray diffraction) don't match other results, there is no issue with that. You should be able to make hypothesis and find references to support it. It will open another path for future research.
*check your citation in the text accordingly to the Journal citation style.
Author Response
Response to Reviewer 3 Comments
Point 1: *Third paragraph from down, add some references to the confused concept of rice pearl and rice chalkiness
Response 1: New references have been added regarding the confusing terms regarding whte-core, white-belly and stress-induced chalkiness
Point 2: * Have you used "Olesa" cultivar in this study ? Why is it present in fig1?
Response 2: PL12 is the code for Olesa cultivar, it is synonimous, PL12 has been replaced for Olesa in the whole document
Point 3: *At what height the rice grain images were captured and what imaging analysis software have you used?
Response 3: It was described in Material and Methods 2.3 section that “The images were captured using a Panasonic Lumix DC-GH5 camera and were digitally processed using the IMAT software developed at the Centres Científics i Tecnològics from Universitat de Barcelona (CCiTUB, Barcelona, Spain). The grain profiles were auto-recognized and the pearl profiles were manually selected. The reported values correspond to the average of 100 grains.”
Point 4: *Title 2.7(Write it all)
Response 4: SDS-Polyacrylamide gel electrophoresis (PAGE) is written now
Point 5: *Results: 3.1 Discuss your results quantitatively. Same comment for 3.5
Response 5: Quantitative data is provided within the 3.1 and 3.5 results section.
Point 6: *Indeed, Lin et al., ......... Check out this sentence
Response 6: The sentence has been improved: “Indeed, Lin et al. [6] reported that the amylose content is not related to the presence of pearl per se”.
Point 7: *Your results (X-Ray diffraction) don't match other results, there is no issue with that. You should be able to make hypothesis and find references to support it. It will open another path for future research.
Response 7: True, we have included this in the results section:
Other authors find peaks exactly in the same degrees [25], being relative intensities lower in stress-induced chalky grains from crystalline varieties than in perfect ones [25]. In our case, Olesa long grained stress-induced chalky grains clearly result in a lower intensity diffraction pattern when compared to normal grains matching the results, while Montsainell pearled variety and Guadiamar crystalline variety behave the opposite. In the other hand, white-belly Carnaroli variety and white-core Bomba pearled variety yield similar intensities between stress-induced chalky and normal grains. That points that the optical properties of stress-induced chalky grains can be similar lower or higher than perfect grains depending on the variety.
And this in the discussion section:
Patindol and Wang studied the degree of crystallinity in perfect and chalky grains of six crystalline cultivars and found that this value was always higher in the chalky grains than in the perfect grains of each cultivar [18]. This premise does not match our results for the white-core Montsianell cultivar and the crystalline Guadiamar cultivar in tendency, while no differences were found for the white-core cultivar Bomba and white-belly cultivar Carnaroli. Interestingly, Olesa crystalline variety behaved the opposite, having statistically significant lower intensity in chalky grains. Patindol and Wang also found an inverse relationship between crystallinity and amylose content, a result that also does not match ours; for example, the highest amylose values belonged to Carnaroli, whereas its crystallinity percentages were not the highest among the cultivars tested [18].
Point 8: *check your citation in the text accordingly to the Journal citation style.
Response 8: The citation in the text is in accordance to MDPI journals citation style.
Point 8: The abstract can be improved. Can be rewritten and focused on the main objective of the work.
Response 8: The abstract has been improved.
