Red light-transmittance bagging promotes carotenoid accumulation of grapefruit during ripening

Light, a crucial environmental signal, is involved in the regulation of secondary metabolites. To understand the mechanism by which light influences carotenoid metabolism, grapefruits were bagged with four types of light-transmitting bags that altered the transmission of solar light. We show that light-transmitting bagging induced changes in carotenoid metabolism during fruit ripening. Compared with natural light, red light (RL)-transmittance treatment significantly increases the total carotenoid content by 62%. Based on weighted gene co-expression network analysis (WGCNA), ‘blue’ and ‘turquoise’ modules are remarkably associated with carotenoid metabolism under different light treatment (p < 0.05). Transcriptome analysis identifies transcription factors (TFs) bHLH128, NAC2-like/21/72, MYB-like, AGL11/AGL61, ERF023/062, WRKY20, SBPlike-7/13 as being involved in the regulation of carotenoid metabolism in response to RL. Under RL treatment, these TFs regulate the accumulation of carotenoids by directly modulating the expression of carotenogenic genes, including GGPPS2, PDS, Z-ISO, ZDS2/7, CRTISO3, CYP97A, CHYB, ZEP2, CCD1-2. Based on these results, a network of the regulation of carotenoid metabolism by light in citrus fruits is preliminarily proposed. These results show that RL treatments have great potential to improve coloration and nutritional quality of citrus fruits.

Line 105-106 "light -----as the crucial medium for the exchange of information between the plant and external environment" is this exactly explain? Line 154 "ζ-carotene lycopene "is "ζ-carotene, lycopene "? Line 226 "metabolism underlying fruit ripening previously had not been elucidated " There are some paper about light effects on fruit quality during development. Line 350-352 "red-light-transmitting bags (RL) (peak wavelength, 748 nm), blue-light-transmitting bags (BL) (peak wavelength, 478 nm), and white-light-transmitting bags (WL) ". From Fig. S1 The transmittance of far-red (peak wavelength, 748 nm) are similar between RL and WL. Due to higher transmittance from 450-748 nm in WL, the effects of bagging treatments on carotenoid in grapefruit may be intergrated effects of light intensity and quality. So the transmitted light spectrum under different bags at same light intensity of sun light source should measured.

Reviewer #2 (Remarks to the Author):
This is an interesting manuscript, however, I feel validation is required of the major conclusions. I appreciate that expression of these gene correlates with enhanced carotenoids content. However, this does not prove that their expression is causal. This would require some form of genetic and or pharmacological experiment targeting the genes and gene products respectively. In the absence of such validation the main conclusion of the manuscript is not sufficiently supported. Besides this major flaw the manuscript is well presented and contains generally well carried out experiments however although the most parsimonious conclusion it definitely requires further support.
Reviewer #3 (Remarks to the Author): Authors of the manuscript no COMMSBIO-21-2202 have performed the transcriptomic and metabolic analysis of 'Huoyan' grapefruit subjected to different light signals during fruit ripening. The aim of the study was to elucidate the role of light quality on the carotenoid accumulation in grapefruit and to understand the transcriptional regulatory mechanism underlying light signals during fruit ripening. The authors showed that the carotenoid accumulation in grapefruit responded differently to light quality, and that the red light have significantly inducing role during fruit ripening. The process was modulated by multiple TFs as well as carotenogenic genes. Based on detected DEGs, carotenoids determination and WGCNA, a preliminary regulatory model of red light-induced carotenoid metabolism in grapefruits was proposed.
Regarding the experimental part of work, the authors have used advanced and suitable methodology. The experiments are rather well designed, carefully performed and seem quite reliable (questionable issues listed below). The information included in tables and figures is clear (apart from issues commented below). The results are appropriately discussed; the novelty of presented work is justified; hypotheses are drawn and the potential for the industrial application is highlighted. Except for minor stylistic and typing mistakes, the manuscript is written with adequate English. The work has a potential to bring novel information to the field of study, however, in its current form it needs a major revision. There are several major and minor issues which should be addressed or explained before considering this work for publishing in Communications Biology.
Major points: 1. Materials and methods; Plant material and treatments section: please, add the information about the permeability of the bags for oxygen and CO2.
2. Materials and methods; Plant material and treatments section: In line 356 you say that "fruits were cut into small cubes". Please, explain exactly, which part (tissue) of the grapefruit was used for each particular analysis. Use botanical terms.
3. Materials and methods; Plant material and treatments section: You have used dark-shading bags (DS) as control conditions in your experiment. Is there any particular reason why a daylight was not included as a control? 4. Materials and methods; Library construction section: Have you estimated the RNA integrity number (RIN) for your RNA samples and was it high enough for the library construction and RNAseq? What was the name of the Illumina kit for the library synthesis/ oligo (dT) beads/ adapters -add names of the products and names of manufacturers.
5. Materials and methods; Library construction section: There is no record of the PRJNA728380 BioProject in the SRA (NCBI) database. You state that eight mRNA libraries were constructed for RNA-seq (4 light treatments x 2 harvest points), each in three biological replicates. That would make 24 libraries sequenced in total. Table S2 and Table S3 does not confirm that and the absence of the BioProject from the SRA database does not allow to verify the information of the number of biological replicates sequenced. Please, comment on that and make the SRA information available to the reviewers and readers. 6. Line 320: should be: "positively correlated" 7. Line 332: should be: "responds" 8. Figure 3: colours of the Venn diagrams are slightly misleading. Try to use stick to the colours of your light treatments and use red, blue, black and grey (for white), or -do not use colours at all. 9. Figure 7. Explain all abbreviations used in the figure in the figure description.
10. The Table S7 is not cited in the main text.
11. Provide the reference number of the grapefruit reference genome which was used for the gene annotation here.
Summarizing, I would recommend presented manuscript for reconsidering after correcting the major and minor issues listed above.

Reviewer #1:
Question 1: Line 108-110 'the majority of studies have focused on the effect of postharvest light treatment on fruit quality, with only a few referring to the impact of developmental light treatment on fruit quality". There are some paper about light effects on fruit quality. The following paper may be the newest one about red light affecting citrus fruit carotenoid. Jinli Gong, Yunliu Zeng, Qiunan Meng, Yajie Guan, Chengyang Li, Hongbin Yang, Yingzi Zhang, Charles Ampomah-Dwamena, Ping Liu, Chuanwu Chen, Xiuxin Deng, Yunjiang Cheng and Pengwei Wang. Red light-induced kumquat fruit coloration is attributable to increased carotenoid metabolism regulated by FcrNAC22 (2021).

Answer:
We accepted your suggestion. the reference was added in the revised manuscript, see reference list No.13 in line 514-516, and the description was modified as "only several works referring to the impact of developmental light treatment on fruit quality" in line 110.
Question 2: Line 105-106 "light-as the crucial medium for the exchange of information between the plant and external environment is this exactly explain? Answer: We are sorry for our unclear description. The sentence was corrected as "as an environmental signal to participate in the regulation of a variety of metabolic processes during plant development". See lines 105-107.
Question 4: Line 226 "metabolism underlying fruit ripening previously had not been elucidated" There are some paper about light effects on fruit quality during development.
Answer: We accepted your suggestion. We modified the sentence as " As a usual agricultural practice in citrus cultivation, the role of light-transmittance bagging in carotenoid metabolism underlying grapefruit ripening previously had remained elucidated." and as is shown in line 234-236.
Question 5: Line 350-352 red-light-transmitting bags (RL) (peak wavelength, 748 nm), blue-light-transmitting bags (BL) (peak wavelength, 478 nm), and whitelight-transmitting bags (WL) From Fig. S1 The transmittance of far-red (peak wavelength, 748 nm) are similar between RL and WL. Due to higher transmittance from 450-748 nm in WL, the effects of bagging treatments on carotenoid in grapefruit may be integrated effects of light intensity and quality. So, the transmitted light spectrum under different bags at same light intensity of sun light source should be measured.
Answer: Thank you for pointing it out. Sure, the same intensities of the light irradiation should be used in the similar study, which seems to be convenient to be compared. In doing so, if the intensity is the same under different light-transmitting bagging, the effect of light on carotenoid will only light quality.
As an effective method of protecting fruit from insect infestations, bird attack, and sunburn as well as reducing disease incidence rate and chemical residues, fruit bagging is extensively used in modern orchards. And the different light-transmitting bags used in the study is the bags widely used in citrus cultivation in China.
However, the protection cultivation and LED should be used for excise control the light intensity of each light quality, the cost is high, it is unbenefited for extension to practice. So, in the study, we only compared the regulation effect of these bags widely used in practice on fruit quality. Our results showed that red-light-transmitting bags (RL) is the optimal bags. It will be used in practice.
According to your comment, the light intensity was completed in Supporting information Fig.1C. In addition, other micro-climate parameters of different light-transmitting bags were also added in the Supporting information Fig.1C. Yes, you are right, the intensity under different light-transmitting bags are different. So the effects of bagging treatments on carotenoid in grapefruit is integrated effects of light intensity and quality. Based on it, the manuscript title was modified as "Transcriptomic and metabolic analysis uncovers the role of red light-transmittance bagging in promoting carotenoid accumulation of grapefruit during ripening". Furthermore, the related discussion was also modified, see lines 364-366.

Reviewer #2 (Remarks to the Author):
This is an interesting manuscript; however, I feel validation is required of the major conclusions. I appreciate that expression of these gene correlates with enhanced carotenoids content. However, this does not prove that their expression is causal. This would require some form of genetic and or pharmacological experiment targeting the genes and gene products respectively. In the absence of such validation the main conclusion of the manuscript is not sufficiently supported. Besides this major flaw the manuscript is well presented and contains generally well carried out experiments however although the most parsimonious conclusion it definitely requires further support.
Answer: Thank you for your comments. The expression of these gene related with carotenoids content and some regulators was validated by qPCR. See the Fig. 7 in line 689-694 in revised manuscript.

Reviewer #3:
Question 1: Materials and methods; Plant material and treatments section: please, add the information about the permeability of the bags for oxygen and CO 2 .
Answer: We accepted the suggestion. the information about the permeability of the bags for oxygen and CO 2 was added in the Supporting information Fig.S1C, and related description was also complemented in the revised manuscript, see lines 364-366.
Question 2: Materials and methods; Plant material and treatments section: In line 356 you say that 'fruits were cut into small cubes'. Please, explain exactly, which part (tissue) of the grapefruit was used for each particular analysis. Use botanical terms.

Answer:
We are very sorry for our unclear descriptions. In fact, only the editable juice vesicles of grapefruits were used for analysis. The related information was added in 370.
Question 3: Materials and methods; Plant material and treatments section: You have used dark-shading bags (DS) as control conditions in your experiment. Is there any particular reason why a daylight was not included as a control?
Answer: Thank you your comments.
In 2020 we used dark as the control. To verify the treatment effect, in 2021 we redo the experiment using natural light (no bagging). As doing so, all 2021 data were used in the revised manuscript. For ensure the consistency the results, all transcriptomic and metabolic data was analyzed using 2021 data. And all graphs were made based on the new analyzed results. See the revised manuscript.
Question 4: Materials and methods; Library construction section: Have you estimated the RNA integrity number (RIN) for your RNA samples and was it high enough for the library construction and RNA-seq? What was the name of the Illumina kit for the library synthesis/ oligo (dT) beads/ adapters -add names of the products and names of manufacturers.
Answer: Thank you for point this out.
We evaluated the RNA integrity number (RIN) for all RNA samples and RIN score ranged from 7.8~9.2, which suggested RNA integrity is qualified (Supplementary  Table S2). Therefore, sample can be used for RNA-seq.
RNA-Seq libraries for maturation and fully ripe-stage grapefruit under CK, RL, BL and WL treatment were constructed using TruePrepTM DNA Library Prep Kit V2 for Illumina® (Vazyme, Nanjing, China) according to manufacturer's manual and seen in line 410-412.
Question 5: Materials and methods; Library construction section: There is no record of the PRJNA728380 BioProject in the SRA (NCBI) database. You state that eight mRNA libraries were constructed for RNA-seq (4 light treatments x 2 harvest points), each in three biological replicates. That would make 24 libraries sequenced in total.
My comments were properly responded. but there were some commmets: Line 253 'ζ-carotene lycopene ' should be 'ζ-carotene, lycopene '. Line 271 'These results showed that red light play important regulatory role in carotenoid metabolism of grapefruit.' These were the results of red light-transmittance bagging, not the results of red light. Line 356 'a preliminary regulatory model of red light-induced carotenoid metabolism' This was the result of red light-transmittance bagging, not the result of red light.
Reviewer #3 (Remarks to the Author): Dear Authors, Compared to its previous version, I find the revised manuscript largely improved. You have professionally addressed all minor and major issues. Especially, I would acknowledge complementing the current manuscript with the data from the natural light experiment as well as with the real-time qPCR analysis. Additionally, I have found very few minor issues, which should be quickly corrected before publication. While reading the revised manuscript, I have spotted several typing mistakes (f.e.: correct into "Relative" in Table 1C or "verification" in line 346), therefore I would encourage the Authors to double-check the manuscript text throughout or use professional proofreading service. The manuscript would certainly benefit from adding the information about the method used for calculation of relative expression levels (in Materials and methods, Real-time quantitative PCR section). Was the Livak method (Livak and Schmittgen, 2001) or the Pfaffl method (Pfaffl, M.W., 2001. A new mathematical model for relative quantification in real-time RTPCR. Nucleic Acids Res. 29, 16-21.) used? Also, as the real-time PCR reactions were performed with the SYBR Green dye, it would be beneficial to add the information about the specificity of real-time PCR reactions (namely: was the melting curve/ dissociation curve analysis done for each amplification product?). Including all the above-mentioned information into your manuscript would certainly further improve it, however, to my opinion the manuscript is ready for publication in its current form.