The content of total polyphenols was never less than 278 μg g−1 of fresh product. Hyperoside, chlorogenic and gallic acids were the most abundant polyphenols. The interrelationships between the parameters analyzed and the different landraces showed that total polyphenols could have mostly affected (ρ = 0.76) the antioxidant activity more than lycopene (ρ = −0.96). The interrelationships between the most abundant polyphenols and antioxidant activity showed that hyperoside, although the most abundant, negatively affected (ρ = −0.93) the antioxidant activity. Due to the high content of lycopene, ascorbic acid, and hyperoside, the five landraces of the tomato “Piennolo” could be considered promising in terms of their potential healthy characteristics.Our study aimed to analyze some qualitative aspects of five landraces of the tomato “Piennolo,” typical of the Vesuvian area, Italy, and determine the in vitro antioxidant activity. All samples showed a high °Brix value and acidity, as well as a discrete amount of reducing sugars, indicating their good quality as fresh products. They showed a high content of lycopene (up to 218 μg g Solanum lycopersicum) constitutes one of the most popular products cultivated worldwide [The current needs of agriculture and agro-industry have induced farms to pursue certain strategies with the aim of increasing productivity and reducing the costs of production to maintain an adequate level of competitiveness , as theyorldwide  and provorldwide . Concurrorldwide . The tomorldwide ,13. In torldwide , severalorldwide ,16. The orldwide . “Piennoorldwide . Therefoorldwide  reportedorldwide  evaluatep-coumaric acid, gallic acid, chlorogenic acid, catechin, epicatechin, hyperoside, rutin, quercetin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), β-carotene, ascorbic acid, HPLC-grade methanol, NaOH, sulphuric, metaphosphoric, acetic and formic acids, acetonitrile, petroleum ether, ethanol and acetone were purchased from Sigma-Aldrich . Apigenin was purchased by Extrasynthese . The Folin–Ciocalteu reagent was purchased by BIO-RAD . Water was distilled and filtered through a Milli-Q apparatus  before use.Caffeic acid, ferulic acid, 2. The irrigation and plant protection as well as the weed control were carried out following local practices. For the experimental design we considered three replicates and 10 plants per replicate. Tomato fruits were harvested based on the same sun exposition, mature ripe stage (according to the full appearance of red color on the fruit surface) and size, and gently cleaned. Peduncles and seeds were removed, some of the fresh fruit was subjected to chemical analysis, and other samples were instantly stored at −30 °C for the biochemical analysis.Five landraces of the tomato “Piennolo”, listed by the Official Bulletin of the Campania Region , were all grown and collected in the farm of the “Cooperativa ARCA 2010” sited in Acerra (NA), Italy. Acerra is characterized by a Mediterranean climate with an average of air temperature (T), humidity (U), and rainy days (R) = 22.7 °C; U = 63.8%; R = 6.6 during the growing season [® 400 Circulator, VWR International Srl, Milano, Italy) for 3 cycles × 1 min and filtered through Whatman N.l filter paper . The residue was discarded, while the filtrate was used for chemical analyses. The refractometric index (°Brix), pH value, titratable acidity, and amount of reducing sugars were determined following the Italian National Official bulletin (1989) for the analysis of vegetable and vegetable-derived products [Frozen diced tomato sample was quickly thawed by placing under running tap water. They were homogenized  and incubated (1:3 w/vol) in methanol (containing acetic acid 1%) overnight at 4 °C. After centrifugation , supernatants were recovered; pellet was treated again with the same extractive solution two times. The three supernatants were pooled, filtered twice , Italy), and the polyphenol amount and profile as well as the antioxidant activity were evaluated. The content of total polyphenols was evaluated at λ = 760 nm , with Folin-Ciocalteau reagent, using gallic acid as standard [−1 of fresh sample.Samples were homogenized . From the upper phase, 2 mL were taken, which was read with a spectrophotometer at 450 nm , using petroleum ether as blank, and with an extinction coefficient ε = 2592. The concentration of β-carotene was established by the standard curve produced with β-carotene standard [The extraction of the carotenoids was done by crushing 2 g of sample in 2 mL of ethanol ; 3 mL of petroleum ether was added to it, and the mixture was vigorously shaken. Each sample was then centrifuged for 15 min at 1000× standard .Samples were crushed and incubated (1:5 w: vol) in a mixture composed of hexane–methanol–acetone (2:1:1 vol:vol:vol) containing 2.5% BHT (butylated-hydroxy-toluene). The supernatant was recovered and read at λ: 520 nm and λ: 450; the first wavelength was used to measure the amount of lycopene, and the second wavelength was used to cancel out the possible interferences of the other carotenoids . From th® 400 Circulator, VWR International Srl, Milano, Italy) and incubated with three volumes of metaphosphoric acid (4%) and kept for 1 h at 4 °C in the dark. Extracts were centrifuged for 10 min at 4 °C and 11,600× g  and filtered . The chromatographic determination of ascorbic acid was performed by liquid chromatography, using an RP-HPLC Gold System chromatograph with a UV detector  and a Khromasil KR 100-5 C18 column (25 cm × 4.6 mm) at room temperature. Sulphuric acid at 0.001 M in HPLC-grade water was the mobile phase used. The injection volume was 20 μL, the flow rate was set at 1.0 mL/min, and the detection wavelength was set at 245 nm. The concentration of ascorbic acid was established by the standard curve produced with ascorbic acid standard solution.The dosage of ascorbic acid was performed following the method of Nazzaro et al. . Briefly−5 M) and measuring the absorbance at λ = 517 nm (Cary 50 MPR). The absorbance of DPPH without antioxidant (control sample) was used for baseline measurements. The scavenging activity was calculated as a percentage. The experiments were performed in triplicate. Results were expressed as the mean values ± standard deviation.The radical-scavenging activity was determined in microplates using the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH assay) . The anaTM system  linked to a PDA 2996 photodiode array detector (Waters) was used for ultra-high-performance liquid chromatography (UPLC) analysis, following the method of Pane et al. [18, 1.7 μm, 2.1 × 100 mm; Waters). The mobile phase consisted of solvent A (7.5 mM acetic acid) and solvent B (acetonitrile) at a flow rate of 250 μL min‒1. A gradient elution was employed, starting with 5% B for 0.8 min, then 5–20% B over 5.2 min, isocratic 20% B for 0.5 min, 2%30% B for 1 min, isocratic 30% B for 0.2 min, 30–50% B over 2.3 min, 50–100% B over 1 min, and isocratic 100% B for 1 min, 5–100% B over 0.5 min, and finally the column was equilibrated under the initial conditions for 2.5 min. The pressure ranged from 6000 to 8000 psi. The injection volume was 5 μL. The effluent was introduced into an LC detector .An ACQUITY Ultra Performance LCe et al. . The acqAs concerns chemical parameters, data were expressed as the mean ± the standard deviation of triplicate measurements; calculations were performed using the PC software “Excel Statistics”. Interrelationships (in terms of linear correlation) between bio-chemicals parameters and antioxidant activity of the different varieties were investigated  by measuThe data for some of the chemical parameters evaluated for the five landraces of the tomato “Piennolo” are shown in From these data, it emerged that, regarding the °Brix value, all samples exhibited a high °Brix ranging between 6.4 (“Principe Borghese”) and 7.6 (“Piennolo di Pollena”). Such values fall perfectly within the range of refractometric values provided, for example, for cherry or cluster tomatoes , which nThe five landraces of “Piennolo”, thanks to a high °Brix, a pH lower than 5.0, and a high natural acidity, might be potentially able to be kept as a fresh product for many months even at room temperature, which is the natural status for this type of tomato, which is generally grouped and hung in a “Piennolo” style and stored outside the balconies and terraces of Naples and of many towns in Campania in general. The data obtained from the analysis could indirectly demonstrate other qualitative parameters for the five ecotypes of “Piennolo”, such as the flavor (determined by a high sugar content) and taste  ,33.In −1 of fresh product (found in the variety “Centoscocche”) and 320 μg g−1 of fresh product, observed in the varieties “Piennolo di Pollena” and “Piennolo Vesuviano”  and 218.89 μg g−1 of fresh product (in the “Centoscocche”). These values were higher than those found, for example, by Carrillo et al. [−1 of fresh product, and found also a level of β-carotene in line with or in some cases lower than those observed in the different landraces of “Piennolo” we analyzed, which showed values of β-carotene ranging between 1.93 μg g−1 of fresh product (“Piennolo del Vesuvio”) and 1.068 μg g−1 of fresh product (“Piennolo Principe Borghese”). The other landraces had a β-carotene content corresponding to 1.34 μg g−1 of fresh product (“Piennolo Centoscocche”), 1.36 μg g−1 of fresh product (“Piennolo Rosso Vesuviano”), and 1.65 μg g−1 of fresh product (“Piennolo di Pollena”). The remarkable antioxidant capacity of lycopene is essentially due to its structural conformation, which allows it to release electrons and thus slow down the oxidation processes that can affect mono and polyunsaturated fatty acids [Akkermansia and also having an impact on obesity [We evaluated the amounts of two carotenoids considered important molecular parameters for the product and to be healthy key molecules, which were β-carotene and lycopene, with this last being the most important carotenoid present in tomatoes. Results are shown in o et al.  in othero et al. , who anaty acids . This prty acids , who monty acids . As a lity acids ,43,44,45ty acids . In rece obesity ,48. Thus−1 of fresh product  to 275 μg g−1 of fresh product (present in the “Principe Borghese”), with intermediate values of 233 μg g−1 of fresh product (in the “Piennolo Rosso Vesuviano”), 238 μg g−1 of fresh product (in the “Piennolo Centoscocche”), and 248 μg g−1 of product (found in the “Piennolo Vesuviano”). These values were also higher than those observed by Caruso et al. [The ascorbic acid (AA) content ranged from 197 μg go et al. , who fouo et al. , Garcia-o et al. , and thoo et al. , which rThe analysis of the polyphenol profile, performed by UPLC, allowed us to recognize and to quantify some metabolites. Results of the analysis are shown in −1 of fresh product) and a variable amount of rutin was found in almost all varieties (except “Piennolo Rosso Vesuviano”), which ranged from 6 μg g−1 of fresh product (“Piennolo Centoscocche”) to 34 μg g−1 of fresh product (discovered in “Piennolo Principe Borghese”). Some of the metabolites identified through UPLC were also identified by Del Giudice et al. [p-coumaric acid and naringenin; on the contrary, we found a large amount of hyperoside , which was the most abundant metabolite among those recognized by the chromatographic analysis, confirming the presence of an abundant amount of flavonoids in tomatoes. The amount of other polyphenols, such as ferulic acid and gallic acid resulted as much higher, with respect to the study carried by Vallverdu et al. [−1 of fresh product, respectively. The identification of ferulic acid but not of p-coumaric acid probably meant that the metabolic pathway did not follow the route toward the formation of lignin but took a “horizontal route”, with the subsequent production, from p-coumaric acid, of ferulic acid [−1 of dry product weight. The same cultivar, however, exhibited an undoubtedly much higher quantity of rutin when compared to our results [Hyperoside, chlorogenic acid, and gallic acid resulted as the most abundant metabolites. We did not find quercetin, apigenin, or epicatechin. Among the flavonoids, we detected also very low amounts of catechin  and less related  to the content of β-carotene (ρ = 0.38) and of ascorbic acid. Lycopene, although present at a high concentration in the tomato landraces, affected in a negative way the antioxidant activity, so to give ρ = −0.96 (with p = 0.01). The higher influence of polyphenols on the antioxidant activity is in agreement with the findings of Ahmed et al. [The landraces of “Piennolo” we analyzed exhibited an antioxidant activity ranging between 17.39% (shown by the “Piennolo Centoscocche”) and 23.91% (which was revealed by the variety “Piennolo Principe Borghese”, d et al.  and Patad et al. . Thus, sp = 6 × 10−7, The analysis of correlation between the antioxidant activity and the most abundant metabolites, hyperoside, chlorogenic and gallic acids, identified and quantified through UPLC, revealed interesting aspects. Regarding hyperoside, the interrelationship between the concentration of this compound in each sample and the antioxidant activity indicated ρ = −0.93 (−1; thus, hyperoside seemed to apply a slightly lower inhibiting effect on the antioxidant activity of “Piennolo Rosso Vesuviano” than on “Piennolo Cento Scocche”. “Piennolo Principe Borghese” and “Piennolo Vesuviano” showed similar antioxidant activity: in this case it could be observed that, compared to total polyphenol content, the amount of hyperoside present in the “Piennolo Vesuviano”, although superior respect to the “Piennolo Principe Borghese” resulted as proportionally inferior. Concurrently, in agreement with Slimestad et al. [From these data and in disagreement with other works  it couldd et al.  and Apakd et al.  chloroge“Piennolo”, like other Mediterranean long storage tomatoes, constitute an important part of local heritage and culture, but it is still commercialized in local markets. Like the other tomatoes with such peculiarity, it was selected since ancient time under the typical Mediterranean climate conditions. Therefore, “Piennolo” tomato could even better respond to the varied climatic conditions, now tending to persist in higher temperatures and a lack of water resources. The high values of °Brix and acidity, as well as high sugar content and low pH, observed in the five landraces of Piennolo allowed us to consider them as products with qualitative valence, for whom it is not necessary to act modifying the chemical parameters to avoid their deterioration, so that they may be consumed both as fresh and as processed products. Furthermore, the high content of lycopene and ascorbic acid, as well as the polyphenols detected, indicate that they could represent, like other varieties of tomato ,59,60,61


Helianthus annuus L.) is one of the important vegetable oil supplies in the world. However, the information about WRKY genes in sunflower is limited. In this study, ninety HaWRKY genes were identified and renamed according to their locations on chromosomes. Further phylogenetic analyses classified them into four main groups including a species-specific WKKY group. Besides, HaWRKY genes within the same group or subgroup generally showed similar exon-intron structures and motif compositions. The gene duplication analysis showed that five pairs of HaWRKY genes  are tandem duplicated and four HaWRKY gene pairs  are also identified as segmental duplication events, indicating that these duplication genes were contribute to the diversity and expansion of HaWRKY gene families. The dN/dS ratio of these duplicated gene pairs were also calculated to understand the evolutionary constraints. In addition, synteny analyses of sunflower WRKY genes provided deep insight to the evolution of HaWRKY genes. Transcriptomic and qRT-PCR analyses of HaWRKY genes displayed distinct expression patterns in different plant tissues, as well as under various abiotic and biotic stresses, which provide a foundation for further functional analyses of these genes. Those functional genes related to stress tolerance and quality improvement could be applied in marker assisted breeding of the crop.WRKY transcription factors play important roles in various physiological processes and stress responses in flowering plants. Sunflower ( WRKY gene family is considered as one of the largest transcription factor (TF) family in higher plants Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The authors conducted a genome-wide analysis of WRKY gene family in sunflower under drought, salt and weed stresses. The genes were characterized by phylogenetic analysis, gene structure and motif analysis, and expression analysis. Synteny analysis of the genes between sunflower and two other species, rice and Arabidopsis, were also done to understand the evolutionary aspects. The article is well-written. I would recommend publication upon minor revisions as indicated below:Line 26: add a comma after “WKKY group”; add “the” before “same group”Line 33: the last sentence could change into ”Those functional genes related to stress tolerance and quality improvement could be applied in marker assisted breeding of the crop”.Line 86: add a comma after “abiotic constraints”Line 92: Asteraceae should not be italic, please change into “Asteraceae family”Line 167-169: the sentence “For biotic stress, common sunflower cultivars TK0409 (susceptible) and JY207 (resistant), and root parasitic weed Orobanche cumana were applied” is not clear. Do you mean: “For biotic stress, root parasitic weed Orobanche cumana were applied to common sunflower cultivars TK0409 (susceptible) and JY207 (resistant)”?Line 194-203: please also indicate if house-keeping genes were used as reference for the qPCR, and if yes, what genes are used?Line 332: please change “23” into “Twenty-three”Line 370: please delete “fortunately”Line 375: please delete “results” at the end of the lineConclusion section: please also indicate here what HaWRKY genes (and/or how many) are the most important for each of the biotic or abiotic stresses.Fig. 4 legend: “On the right side, Different motifs…” please change “Different” into “different”.Reviewer #2: Comment of ReviewerIn the manuscript “Genome-wide characterization of WRKY gene family in Helianthus annuus L. and their expression profiles under biotic and abiotic stresses”, Li et al. identified WRKY family genes in sunflower and renamed them according to their locations on 25 chromosomes. They classified them into four main groups including a species-specific WKKY group using phylogenetic analyses, and they provided deep insight to the evolution of HaWRKY genes in sunflower by synteny analyses. Through transcriptomic and qRT-PCR analyses, they displayed distinct expression patterns of HaWRKY genes in different tissues, and under various abiotic and biotic stresses, which provide a foundation for further functional analyses of these genes.This manuscript provides compelling evidence that HaWRKY family genes are associated with the development of the sunflower, as well as various abiotic and biotic stresses responsive, and provides a foundation for further functional analyses of HaWRKY genes. By using a non-standard genetic model like sunflower, the authors also help diversify studies in plant development. Please find my comments on the manuscript below.1. Abstract is generally well-written but lacks some important data of the results.2. Figure 7 and 8: Student’s t-test is fine here, but I suggest using Tukey’s HSD test to test all comparisons. This will help to show if 10% and 20% PEG, as well as 15mM and 30mM NaCl lines are different from each other, rather than just comparing to CK.3. Line 53 “Functional analyses show that WRKY…” should be “Functional analyses showed that WRKY g…”.4. In Materials and Methods part, some paragraph should could be more concise such as plant materials, growth conditions and treatments section.5. The English writing should be substantially improved. Many sentences are illogical and cannot be understandable.6. Overall: Add test statistics and p-values where significance is mentioned in text.7. Overall: Italicize gene names.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  20 Oct 2020Responses addressing Reviewers’ CommentsComments of Reviewer #1: The authors conducted a genome-wide analysis of WRKY gene family in sunflower under drought, salt and weed stresses. The genes were characterized by phylogenetic analysis, gene structure and motif analysis, and expression analysis. Synteny analysis of the genes between sunflower and two other species, rice and Arabidopsis, were also done to understand the evolutionary aspects. The article is well-written. I would recommend publication upon minor revisions as indicated below:Detailed comments:Remark: Line 26: add a comma after “WKKY group”; add “the” before “same group”Answer: Thanks for your kind comments. We have added a full stop after “WKKY group”; and added “the” before “same group” in Line 28. We also have tried our best to improve the Abstract Section in red front.Remark: Line 33: the last sentence could change into “Those functional genes related to stress tolerance and quality improvement could be applied in marker assisted breeding of the crop”.Answer: Many thanks for your kind suggestion. The last sentence has been changed into “Those functional genes related to stress tolerance and quality improvement could be applied in marker assisted breeding of the crop” expression in Line 41-42.Remark: Line 86: add a comma after “abiotic constraints”Answer: Thanks for your kind suggestion. We have added a comma after “abiotic constraints” in Line 93, and added “Moreover,” after the comma to improve the English.Remark: Line 92: Asteraceae should not be italic, please change into “Asteraceae family”Answer: Thank you for your kind comment. In the revised manuscript, “Asteraceae” has been changed into “Asteraceae family” in Line 99 in red font. Moreover, the italic “Asteraceae” was also changed into “Asteraceae” in Line 387.Remark: Line 167-169: the sentence “For biotic stress, common sunflower cultivars TK0409 (susceptible) and JY207 (resistant), and root parasitic weed Orobanche cumana were applied” is not clear. Do you mean: “For biotic stress, root parasitic weed Orobanche cumana were applied to common sunflower cultivars TK0409 (susceptible) and JY207 (resistant)”?Answer: Thanks for your kind suggestion. We have changed this sentence into “For biotic stress, root parasitic weed Orobanche cumana were applied to common sunflower cultivars TK0409 (susceptible) and JY207 (resistant)” in Line 169-171.Remark: Line 194-203: please also indicate if house-keeping genes were used as reference for the qPCR, and if yes, what genes are used?Answer: Thanks for your kind comment. ACT1, a house-keeping gene was used as reference for the qPCR. In the revised manuscript, we have added “and the ACT1 was selected as the reference gene.” after “The 2−ΔΔCt method with three replications was performed for analysis” in Line 202-203. We also have revised accordingly in the Supplementary Table S1.Remark: Line 332: please change “23” into “Twenty-three”Answer: Thanks for your kind suggestion. We have changed the number “23” into “Twenty-three” in Line 334.Remark: Line 370: please delete “fortunately”Answer: Thanks for your kind suggestion. We deleted “fortunately” in Line 378 between “and” and “we”.Remark: Line 375: please delete “results” at the end of the lineAnswer: Thanks for your kind suggestion. We deleted “results” at the end of Line 383.Remark: Conclusion section: please also indicate here what HaWRKY genes (and/or how many) are the most important for each of the biotic or abiotic stresses.Answer: Thank you for your kind comment. In the revised manuscript, we have tried our best to make the conclusion more specific. The sentence “The expressions of HaWRKY genes suggested that these genes could be involved in the regulation of sunflower growth and development, as well as various abiotic and biotic stresses” has been changed into “The expressions of HaWRKY genes suggested that most of the 23 selected HaWRKY genes were found to play an important role in regulating PEG and salt stress and 9 genes including HaWRKY 7/15/44/45/68/71/72/76/85 from 90 HaWRKY genes have potential roles against O. cumana infection” in Line 495-498.Remark: Fig. 4 legend: “On the right side, Different motifs…” please change “Different” into “different”.Answer: Thanks for your kind comment. The legend of Fig. 4 “On the right side, Different motifs…” has been changed into “On the right side, different motifs…”.Comments of Reviewer #2: In the manuscript “Genome-wide characterization of WRKY gene family in Helianthus annuus L. and their expression profiles under biotic and abiotic stresses”, Li et al. identified WRKY family genes in sunflower and renamed them according to their locations on 17 chromosomes. They classified them into four main groups including a species-specific WKKY group using phylogenetic analyses, and they provided deep insight to the evolution of HaWRKY genes in sunflower by synteny analyses. Through transcriptomic and qRT-PCR analyses, they displayed distinct expression patterns of HaWRKY genes in different tissues, and under various abiotic and biotic stresses, which provide a foundation for further functional analyses of these genes.This manuscript provides compelling evidence that HaWRKY family genes are associated with the development of the sunflower, as well as various abiotic and biotic stresses responsive, and provides a foundation for further functional analyses of HaWRKY genes. By using a non-standard genetic model like sunflower, the authors also help diversify studies in plant development. Please find my comments on the manuscript below.Remark 1: Abstract is generally well-written but lacks some important data of the results.Answer: Thanks for your kind suggestion. We have added the important results in the Abstract Section such as “Sunflower (Helianthus annuus L.) is one of the important vegetable oil supplies in the world.” In Line 23-24, and “The dN/dS ratio of these duplicated gene pairs were also calculated to understand the evolutionary constraints.” In Line 35-36.We also have revised “The tandem and segmental duplication possibly contributed to the diversity and expansion of HaWRKY gene families” into “The gene duplication analysis showed that five pairs of HaWRKY genes  are tandem duplicated and four HaWRKY gene pairs  are also identified as segmental duplication events, indicating that these duplication genes were contribute to the diversity and expansion of HaWRKY gene families in Line 30-35. Remark 2: Figure 7 and 8: Student’s t-test is fine here, but I suggest using Tukey’s HSD test to test all comparisons. This will help to show if 10% and 20% PEG, as well as 15mM and 30mM NaCl lines are different from each other, rather than just comparing to CK.Answer: Thanks for your kind comment. In the revised manuscript, we have reanalyzed the significant expression levels of different HaWRKY genes under different treatments carefully by Tukey’s HSD test in Fig. 7 and Fig. 8. It is important to test all comparisons, especially for different concentrations of PEG and NaCl treatments. We also have changed “Student’s t-test” into “Tukey’s HSD test” in Materials and Methods section in Line 204.Remark 3: Line 53 “Functional analyses show that WRKY…” should be “Functional analyses showed that WRKY g…”.Answer: Thanks for your kind suggestion. The sentence “Functional analyses show that WRKY…” has been changed into “Functional analyses showed that WRKY g…”in Line 60.Remark 4: In Materials and Methods part, some paragraph could be more concise such as plant materials, growth conditions and treatments section.Answer: Thanks for your kind comment. We have revised “The seeds were germinated and grown in peat moss. At the five-leaf stage, seedlings with uniform size were selected for hydroponics in a half-strength Hoagland nutrient solution (pH=6.0). ” into “The seeds were germinated and grown in peat moss according to our previous study [32].” in Line 162 and cited the related literature. We also changed “The treatment concentrations were selected based on our preliminary experiments, in which 0, 100, 200, 300, 400 and 600 mM of NaCl were used. Little damage, significant damage and plant death were observed when the concentration of NaCl reached 100 mM, 300 mM, and over 300 mM, respectively. The concentrations of simulated drought stress were selected similarly.” into “The treatment concentrations were selected based on our preliminary experiments [32].” in Line 165-166.Remark 5: The English writing should be substantially improved. Many sentences are illogical and cannot be understandable.Answer: Thanks for your kind suggestion. We have tried our best to improve the manuscript. We revised “Data were transformed with a log2 (FPKM+1) transformation” into “Data were transformed by log2 (FPKM+1)” in Line 186. We deleted “in this WKKY group” between “distributed genes” and “into group I, II and III” in Line 224.We added “ten” between “all” and “tissues” in Line 320, which could make the expression much more specific. We have substantially improved the manuscript, especially the Results section in Line 339-354, which will make the manuscript much more clear and understandable.We also have changed “We identified 90 WRKY genes from Helianthus annuus L. and they were classified into four main groups including a species-specific WKKY group.” into “In this study, we identified 90 WRKY genes from Helianthus annuus L. and characterized their structure, duplication, chromosomal distribution, phylogenetic tree, followed by tissue-differential gene expression and differential expression in response to biotic and abiotic stress.” in Line 489-492.We have revised “This study provides a foundation for further functional analyses of HaWRKY genes” into “Taken together, this study provides a good basis for further investigation of the biological functions and evolution of HaWRKY genes” in Line 498-499 in Conclusion section.For detail, please refer to the revised manuscript in red front.Remark 6: Overall: Add test statistics and p-values where significance is mentioned in text.Answer: Thanks for your kind comment. We have added the test statistics and p-values where significance is mentioned in text. We changed “significantly up-regulated” into “significantly (P < 0.05) up-regulated by 46%/140% and 70%/51% compared with control, respectively.” In Line 339-340. We revised “no any changes of HaWRKY55/57 were observed in response to abiotic stresses” into “But no any significant  changes of HaWRKY55/57 were observed in response to abiotic stresses in all comparisons” in Line 340-341. We also changed “among them HaWRKY22/29/74 were significantly down-regulated only at high concentration of PEG. HaWRKY57/59/77/81/82/89 were significantly induced after treatment with PEG and HaWRKY30/35/38/48/55 were not sensitive to PEG in the roots.” into “For example, HaWRKY13/14/16/57/77 were significantly (P < 0.05) down-regulated under 10% and 20% drought stress, while HaWRKY29/74 were significantly (P < 0.05) down-regulated by 55% and 70% only at high concentration of PEG, as compared with control. The expression level of HaWRKY52 and HaWRKY89 were up-regulated by PEG, and 10% PEG significantly (P < 0.05) increased by 152% and 179% compared with control” in Line 343-349.Remark 7: Overall: Italicize gene names.Answer: Thanks for your kind suggestion. We have carefully checked all name of genes and italicized gene names in Line 336, 462, 573, 580, 583, 593, 619, 676, 678, and Line 683. We also have revised gene names accordingly in the Table 1 captions and the Supplementary Table S1 according to your suggestion. 26 Oct 2020Genome-wide characterization of WRKY gene family in Helianthus annuus L. and their expression profiles under biotic and abiotic stressesPONE-D-20-29683R1Dear Dr. Ling XU,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Basharat Ali, Ph.DAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: All comments have been addressedReviewer #2: All comments have been addressed**********2. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: YesReviewer #2: Yes**********4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********5. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********6. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The authors have addressed all my review comments. I would recommend that the current version is ready for publication.Reviewer #2: (No Response)**********what does this mean?). If published, this will include your full peer review and any attached files.7. PLOS authors have the option to publish the peer review history of their article (If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.Yes: Hui LiuReviewer #1: Reviewer #2: No 17 Nov 2020PONE-D-20-29683R1 WRKY gene family in Helianthus annuus L. and their expression profiles under biotic and abiotic stresses Genome-wide characterization of Dear Dr. Xu:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Basharat Ali Academic EditorPLOS ONE


Genome structural variations (SVs) have been associated with key traits in a wide range of agronomically important species; however, SV profiles of peach and their functional impacts remain largely unexplored.Prupe.4G186800, which encodes a NAC transcription factor, is shown to be associated with early fruit maturity, and a 487-bp deletion in the promoter of PpMYB10.1 is associated with flesh color around the stone. In addition, a 1.67 Mb inversion is highly associated with fruit shape, and a gene adjacent to the inversion breakpoint, PpOFP1, regulates flat shape formation.Here, we present an integrated map of 202,273 SVs from 336 peach genomes. A substantial number of SVs have been selected during peach domestication and improvement, which together affect 2268 genes. Genome-wide association studies of 26 agronomic traits using these SVs identify a number of candidate causal variants. A 9-bp insertion in The integrated peach SV map and the identified candidate genes and variants represent valuable resources for future genomic research and breeding in peach. Solanum lycopersicum) [Glycine max) [Cucumis sativus) [Prunus persica) [Structural variations (SVs), comprising insertions/deletions (indels), duplications, inversions, and translocations, are widely present in genomes –4. In huersicum) , nematodine max) , reprodusativus) , asexualsativus) , and frupersica) . Recent persica) , 12.http://faostat.fao.org) and is regarded as a model plant for the Rosaceae family, thanks to its small genome and relatively short juvenile period [Peach is the fourth largest deciduous fruit crop in the world  technologies and limitations of current computational algorithms can restrain the accuracy of SV detection , 28. To SVs are known to have a major influence on genomes and are often associated with specific traits . We inveDuring peach domestication and improvement, many genetic loci have been selected, and a number of studies have characterized this phenomenon using SNP data , 20. In To further characterize functions of the SVs in peach, GWAS were performed for 26 key agronomic traits, including 18 traits for which GWAS has not been previously reported in peach. Performing GWAS with SNPs is an established methodology and one that takes advantage of the fact that SNPs provide a much higher density of polymorphisms than SVs and can be mapped to a relatively accurate genomic interval , 33–35. MYB gene (Prupe.5G196100) was previously reported to be associated with the fruit hairiness trait, resulting in the peach and nectarine phenotypes [CCD4 gene (Prupe.1G255500) on chromosome 1 have been implicated: a TE insertion, a simple sequence repeat (SSR), and a SNP [Prupe.3G200700 was found to be related to the pendulous branch trait  [We selected 8 qualitative traits that have been targeted in previous GWAS analyses using SNPs , 20 and enotypes , and thind a SNP . Since m Prupe.5G96100 wasa napus) .There is considerable evidence that SVs control many phenotypic traits , 36, 37,Prupe.4G186800, encoding a NAC transcription factor, was reported to be responsible for the MD trait in peach, and a 9-bp insertion was identified as the possible causal variant [Prupe.4G186800 was also detected as the most significant signal in the GWAS analysis  were in the same linkage disequilibrium (LD) block  and its bud mutant “Jinlei” (JL), whose fruit matures 15 days earlier than “SS,” and the “NJC83” cultivar and its bud mutation “Huihuang” (HH), whose fruit matures 10 days earlier, were subjected to comparative transcriptome analysis of fruit development. The differentially expressed genes (DEGs) in a 200-kb genome interval around the candidate region were analyzed, and Prupe.4G186800 was the only gene with much higher expression in both bud mutants  is a critical factor in fruit marketing, and previous studies using linkage analysis have placed the MD locus on linkage group 4 in peach , 41. A psis Fig. a; howeveock Fig. a. By genock Fig. b. To valPpMYB10.1, while more than one gene, but including PpMYB10.1, have been associated with regulating flesh color [PpMYB10.1 promoter region was found to be associated with this trait , a member of which is the key gene controlling fruit shape in tomato [PpOFP1. Phylogenetic analysis of OFP genes in peach and tomato showed a close relationship between PpOFP1 and SlOFP20, a gene known to control tomato fruit shape , including 13 wild, 20 ornamental, 70 landrace, and 233 improved accessions , flesh color around the stone, flower shape , pendulous branch, pollen sterility, hypanthium color (white/yellow), anther color, and kernel taste, were measured in two successive years according to previous reported evaluation criteria  from 201Raw Illumina reads were processed to remove adapter and low-quality sequences. The paired-end reads were mapped to the reference peach “Lovell” genome  (releasehttps://software.broadinstitute.org/gatk/best-practices/). The detection was performed using the GATK HaplotypeCaller, and genotyping was done with GenotypeGVCFs [SNP and small indel calling were performed using the GATK Best-Practices pipeline  was used to generate PacBio long reads for six accessions: “Xinjiang Pan Tao #2,” “Hakuho,” “Tianjin Shui Mi,” “Ying Ge Tao,” “Okubo,” and “Tian Ren Tao.” The PacBio reads were mapped to the reference peach genome using NGMLR  . SVs with significantly different frequencies (FDR < 0.001 and fold change > 2) were identified as those under selection. These SVs were then analyzed to identify the associated genes, and GO enrichment analysis was performed for these genes using the AgriGO [According to the genome annotation, we assessed the putative SV impacts on gene, CDS, and promoter regions according to SV locations and sizes. We set an interval of 2 kb upstream of the gene as the promoter region. To evaluate the SV significance in peach evolution, the occurrence frequencies of each SV were calculated in wild, landrace and improved groups. SVs with higher or lower occurrence frequencies in landrace compared to wild, and improved compared to landrace, were designated as showing positive or negative selection, respectively, during domestication or improvement. The significance of the difference of the frequencies for each SV between the two compared groups (wild versus landrace for domestication and landrace versus improved for improvement) was determined using the Fisher exact test with the R package “pdrtool.” The resulting raw e AgriGO  with a cTo identify candidate genes responsible for the various agronomic traits, GWAS were performed for 26 agronomic traits. We aimed to identify variants associated with these traits by performing GWAS with the SV dataset and the SNP dataset, separately. To improve the accuracy of the GWAS results, we filtered the SV and SNP datasets by removing those with minor allele frequency (MAF) < 0.01. GWAS were performed using MLM, CMLM, and FarmCPU models implemented in GAPIT   samples were synthesized using a cDNA Synthesis System kit  following the manufacturer’s protocol. The qRT-PCR was performed using a LightCycler 480 (Roche) with the following cycle conditions: 95 °C for 5 min, followed by 45 cycles at 95 °C for 10 s, 60 °C for 10 s and 72 °C for 20 s. The house-keeping gene RP-II (RNA polymerase II) was used as an internal control for peach [−ΔΔCt method [To investigate the fruit maturity and flesh color around the stone traits, fruit samples were collected at five stages from “Hakuho”  and “Tianjin Shui Mi” : 20, 40, 60, 80, and 90 DAFB (days after full bloom). For the fruit shape trait, fruit samples were collected at 16 stages from pre-bloom bud to fruit maturation period from “Zhong Tao Hong Yu”  and “Zhong Pan Tao #10” . These samples were used to analyze candidate gene expression patterns. Samples from another two accessions, “Zao Huang Pao Tao”  and “Xinjiang Pan Tao #2” , were collected at 0, 7, 15, and 30 DAFB, to further validate the candidate genes. Total RNA was extracted from these fruit samples using an RNA-extracting kit . To check the relative expression levels of or peach . The relt method .PpOFP1 was amplified from fruit cDNA sample of “XJ2” (aborting flat peach with homozygous genotype) by PCR using a high fidelity DNA polymerase . The products were cloned into the pBI121 vector downstream of the cauliflower mosaic virus (CaMV) 35S promoter using a one-step construction kit . The constructs were then transformed into Micro-Tom tomato using Agrobacterium tumefaciens GV3101 following protocol described in Sun et al. [The full-length coding region of n et al. . After tPpOFP1 were collected at 0 and 10 DAFB and fixed in FAA immediately for 2 days. The samples were then removed from FAA, rinsed thoroughly in deionized water, and then dehydrated using an ethanol gradient, cleared using xylene, and embedded in wax. The embedded samples were then sectioned (10 μm section thickness), and the sections were rehydrated and stained using aqueous toluidine blue (pH 7.0) [Flower and fruit samples of transgenic tomato lines overexpressing (pH 7.0) . Images PpMYB10.1 affects the promoter activity, dual-luciferase reporter assays were carried out. Different promoter regions of PpMYB10.1 were cloned into pGreen-II-0800-LUC vector using the one-step construction kit as described above . The promoters of PpMYB10.1 with or without the deletion were cloned into pGreen-II-0800-LUC to check the influence on gene expression. The constructs were transformed into GV3101 and transient expression assays were performed using tobacco leaves. The relative luciferase activities were detected and photos taken by a Tanon-5200Multi machine .To determine whether the deletion in the promoter region of Additional file 1. Supplementary tables S1-S13.Additional file 2. Supplementary figures S1-S38.Additional file 3. Review history.


Several standard Nc dilution curves were proposed and validated for several crops, based on experiments involving different N fertilizer treatments. However, standard Nc dilution curves are affected by crop water status, e.g., resulting from differences in irrigation management. This paper aimed at developing a N diagnostic model under the coupling effect of irrigation and fertilizer managements. For this purpose, Nc dilution curves were developed under different irrigation rates. Additionally, plant water content (PWC), leaf water content (LWC), leaf area index (LAI), equivalent water thickness (EWT), and leaf area duration (LAD) were introduced into the model, to construct a modified Nc (mNc) dilution curve. The mNc dilution curves were designed using the principle of hierarchical linear model (HLM), introducing aboveground dry biomass (AGB) as the first layer of information, whereas the second layer of information included the different agronomic variables . The results showed that parameters “a” and “b” of the standard Nc dilution curves ranged from 5.17 to 6.52 and −0.69 to −0.38 respectively. Parameter “a” was easily affected by different management conditions. The performance of standard Nc dilution models obtained by the cross-validation method was worse than that of mNc dilution models. The Nc dilution curve based on 4 years of data was described by the negative power equation Nc = 5.05 × AGB–0.47, with R2 and nRMSE of 0.63 and 0.21, respectively. The mNc dilution curve considers different treatments and was represented by the equation mNc = a×AGBb−, where a = 2.09 × PWC + 3.24, b = −0.02 × LAI + 0.51, with R2 and nRMSE of 0.79 and 0.13, respectively. For winter wheat, C3 crop, there would be a few problems in using standard Nc dilution methods to guide field management, however, this study provides a reliable method for constructing mNc dilution curves under different water and N fertilizer management. Due to the significant differences in hereditary, CO2 fixation efficiency and N metabolism pathways for C3 and C4 crops, the construction of mNc dilution curve suitable for different N response mechanisms will be conducive to the sustainable N management in crop plants.The accuracy of nitrogen (N) diagnosis is essential to improve N use efficiency. The standard critical N concentration (standard N Nitrogen (N) is the element used in the largest quantity, as chemical fertilizer in agricultural production, causing environmental concerns worldwide . China ic) dilution curve has been widely used to diagnose the N status of crops (c is defined as the minimum N concentration required to achieve maximum growth at a certain aboveground dry biomass (AGB) accumulation, which is expressed as Nc = a × AGB–b, where the parameter “a” represents standard Nc for per 1 Mg AGB ha–1, and parameter “b” is a statistical parameter governing the slope of the relationship (c-AGB)   and another was for C4 plants (Nc = 4.1 × AGB–0.5: sorghum and maize). Dry matter partitioning among different plant organs affects the shape of the standard Nc curve  , have bec-stage) , or the c-stage) . As a pr has been introduced for the analysis of hierarchically structured data. It is a statistically sound methodology with regression models that explicitly take into account variability at different levels . The modLeaf or stem water potential (ψ) , crop wac dilution model for winter wheat, (ii) to construct modified Nc (mNc) dilution models that include different agronomic variables, i.e., PWC, LWC, LAI, equivalent water thickness (EWT), and leaf area duration (LAD), as correction factors; (iii) to validate the standard Nc dilution model and the newly proposed mNc dilution models using independent data.Our objectives in this study were: (i) to investigate the effects of different irrigation treatments on the standard N–1 organic matter, 8.21 ∼ 41.71 mg kg–1 nitrate-N, 0.45 ∼ 8.20 mg kg–1 ammonium-N, 60.58 ∼ 109.61 mg kg–1 available potassium, and 3.14 ∼ 21.18 mg kg–1 available phosphorus at 0 ∼ 30 cm soil depth. Precipitation data were obtained from the European Center for Medium-Range Weather Forecasts (ECMWF)1  in Beijing, China. The soil was classified as heavy loam  with 15.(ECMWF)1 . The ERA(ECMWF)1 .–1 (N1); (ii) 110 kg N ha–1 (N2); (iii) 220 kg N ha–1 (N3), and (iv) 440 kg N ha–1 (N4). Experiments 2 and 3 were conducted during the 2013 ∼ 2014 and 2014 ∼ 2015 growing seasons, using an orthogonal design of L16(34), with three replicates and testing three factors: N fertilizer rates, wheat varieties, and irrigation rates. The N fertilizer treatments were as follows: (i) 0 kg N ha–1 (N1); (ii) 90 kg N ha–1 (N2); (iii) 180 kg N ha–1 (N3), and (iv) 270 kg N ha–1 (N4). Irrigation rates in experiment 2 were 0 mm (W0), 146 mm (W1), and 292 mm (W2). Irrigation rates in experiment 3 included 0 mm (W0), 192 mm (W1), and 384 mm (W2). Experiment 4 was conducted during the 2015 ∼ 2016 growing season, using a randomized complete block design with three replicates. N fertilizer was applied with the following rates: (i) 0 kg N ha–1 (N1), (ii) 90 kg N ha–1 (N2), (iii) 180 kg N ha–1 (N3), and (iv) 270 kg N ha–1 (N4). Wheat varieties included Lunxuan 167 and Jingdong 18. Fertilizers included urea (46% of N), superphosphate (12% of P2O5), and potassium sulfate (50% of KCl). The N fertilizer was applied in two splits for all experiments: 50% as a basal application before sowing and 50% at the jointing stage. For all treatments, sufficient P (45 kg P2O5 ha–1) and K (75 kg KCl ha–1) fertilizers were incorporated into the soil before sowing. Plot designs for the different growing seasons are inconsistent with Data were obtained from four winter wheat growing seasons, as summarized in Twenty tillers of winter wheat plants per experimental plot were randomly sampled at jointing, booting, anthesis and filling stages, i.e., respectively, Zadoks stage (ZS) 31, 47, 65, 75 , to detefresh, AGB, AGBl–fresh, and AGBl represent the fresh plant aboveground biomass, aboveground dry biomass, fresh leaf biomass and dry leaf biomass, respectively.Where AGBThe N content of leaves, stems, and spikes were determined using the standard Kjeldahl method. PNC was calculated using the following formula:l, Ns, and Np represent leaf N concentration, stem N concentration, and panicles N concentration, respectively. AGBl, AGBs, and AGBp represent dry leaf biomass, dry stem biomass, and dry panicles biomass, respectively.Where NLeaf area was measured with a laser leaf area meter , and the calculation formula of LAI is as follows:m, n, T, and A represent the leaf area of collected wheat, the number of collected tillers, the number of tillers in the survey area and the size of the survey area, respectively.Where LAThe calculation formula of leaf EWT and LAD is as follows:Where T and LAI represent time and leaf area index.c dilution curve approach, it is essential to determine standard Nc points at which N fertilizer neither limits plant growth nor enhances it. We used the same computational procedures followed by c point was determined as follows: (a) the AGB and PNC under different applied N rates were compared through the least significant difference test (LSD) at the 95% level of significance . The N-limiting treatment was defined as a treatment in which an additional N fertilizer application led to a statistically significant increase in the AGB. The non-N-limiting treatment was identified as a treatment in which an additional N fertilizer application did not lead to a significant increase in the AGB, but resulted in a significant increase in the PNC. The AGB and PNC were used to determine whether N treatments limited crop growth. A simple linear regression was utilized to fit data from the N-limiting treatments. For each sampling period, a critical point was defined as follows: (a) the data of AGB and PNC were used to identify if a N treatment limited the growth of the crop; (b) a simple linear regression was used to fit data from the N-limiting treatments (the oblique line); (c) The maximum AGB was calculated with data from the non-N-limiting treatments as the average of the observed data ; (d) the Nc point corresponded to the ordinate of the intersection point of the oblique and vertical lines. c point selection procedure. The Nc points were then fitted using a power regression equation to construct the standard Nc curve. The standard Nc dilution curve is calculated as:With the standard Na” and “b” are parameters obtained by calibrating the standard Nc dilution curve.Where “a” and “b” are considered as constant, regardless of different irrigation and fertilizer treatments, although it is known that these factors influence the parameters  that again considered parameter “a” and “b” as a function of agronomic variables. Therefore, the parameters to be estimated for mNc dilution curve are the four parameters in Level-2 . The HLM model parameters were obtained using optimization algorithm in Matlab 2016  programming. The mNc dilution curve is described as follows:The hierarchical linear model (HLM) was introduced for the analysis of nested structured data . It is a1, β2, β3, and β4 are the parameters to be estimated of the mNc model, respectively. β1 and β3 represent slopes of each agronomic variable. β1 and β3 represent the intercept. AC represents one of the different agronomic variables tested. AC1 and AC2 are one of five agronomic variables  randomly selected, respectively, including 25 combinations. c and the mNc models.Where βc value for the corresponding biomass value. In order to verify the N diagnostic function of mNc dilution curve, the corresponding prediction NNI was calculated as:NNI is the ratio of the actual plant N concentration to the mNc represent nitrogen nutrition index, plant N concentration and predicted critical nitrogen concentration points using the mNc method, respectively.where NNI, PNC, and mNF-test . We evaluated model performance using the determination coefficient (R2) and the normalized root mean squared error (nRMSE). These were calculated as follows:To test whether there was a significant difference in the regression relationships under different management conditions, we examined the difference of regression coefficients using the n, Yi, and p represented the number of samples, predicted value, measured value and the number of independent variables, respectively.Where, c dilution curve, in which more parameters are included, performed better than the standard Nc dilution curve. The AIC value is closely related to L and K value. Among them, the smaller the K value is, the more concise the model is, and the larger the L value is, the more accurate the model is. The general equation is (Akaike information criterion (AIC) was used to find out whether the mNation is :Where L and K represent log-likelihood function and the numbers of estimated parameters, respectively. The PI, Sse and n represent circular constant, residual sum of squares and the samples number, respectively.c dilution theory, the Nc dilution curve divides the data points into three categories: N-limited status below the curve, N-excess states above the curve, and N optimum close to the curve. The average standard Nc dilution curve based on all the data pooled together could be characterized by the negative power equation Nc = 5.05×AGB–0.47, with R2 and nRMSE, of 0.63 and 0.21. However, using the standard Nc dilution curve, there was a large variability depending on the year and water availability conditions  data was significantly different from that derived from all the other experiments. In general, whereas parameter “a” was significantly different among the experiments, there was no significant difference in parameter “b.”Instead, when specific Nre found . The parto −0.38 . StandarP-values) between separate management on the agronomic variables considered are reported in P < 0.05) influence of management on LAI at jointing, booting and anthesis stages under different irrigation and N rates, but not in the filling stage. There was no significant influence of irrigation or fertilization treatments on PWC, LWC, EWT, and LAD.In order to further analyze the coupled effect of irrigation and fertilizer management on different agricultural variables, we examined their differences in different growth periods under irrigation and fertilizer management, using the LSD methodology. The results of the differences  condition, but underestimated predicted Nc value in 2012 ∼ 2013. The Nc dilution curves constructed in this paper under different irrigation conditions were all a negative power type, but there were some differences among the parameters. Parameter “a” and “b” under different treatment were dissimilar. As shown in a” of different management had a significant difference. There was no significant difference for parameter “b” under most treatments. Therefore, different management was shown to affect mainly parameter “a”. The reason for the above results maybe that parameter “a” represents standard Nc for 1 t AGB ha–1. This biomass value might be occurring at different stages for different irrigation treatments. Instead, parameter “b” is a statistical parameter governing the slope of the relationship (c dilution curve could be inaccurate for field management under the coupling effect of water and N fertilizer. The validation results in this paper also proved these problems.on model  overestitionship . While ationship . Therefoc dilution model under different N and irrigation management using different agronomic variables. Plant water provides a medium for enzymatic reactions and is also the raw material of photosynthesis. Plant water directly or indirectly affects the accumulation of plant material (c dilution curve as a function of LAI efficiently identified the N-limiting and non-N-limiting conditions. In addition, through the analysis of variance of multiple factors, different irrigation and fertilization treatments have a significant influence on LAI, but not on PWC. These results were similar to the results of significant differences of parameter “a” and parameter “b.” Therefore, it is feasible to express parameter “a” and parameter “b” using PWC and LAI, respectively. Both parameters are affected concurrently by water and N and show a decline ontogenically (c dilution curve across years and experiments. Moreover LAI and PWC can be readily estimated from remote sensing (This paper proposed a standard mNmaterial . In addienically , so they sensing , it has c dilution model created in this study has more advantages in N diagnosis than standard LAI dilution model. In this paper, C3 crop, which has different N metabolism pathway and characteristics from C4 crop (c dilution curves of C3 and C4 counstructed by c dilution curve by combining N absorption, transport and signal transduction functions and regulatory mechanisms. This study was based on field-scale agronomic data for analysis and verification and did not address mNc dilution curve over considerable areas. In order to establish a real-time, widely-applicable LAI diagnosis model, an appropriate N diagnosis model should be established under multiple cultivation modes, data sources, and scales.The accuracy of nitrogen diagnosis is vital to improve N use efficiency. The mN C4 crop , the stac dilution curve by integrating the effect of water and N fertilizer. Under different management conditions, parameter “a” is more affected than “b.” The mNc dilution curve considers different treatments and is represented by the equation mNc = a×AGBb−, where a = 2.09 × PWC + 3.24, b = −0.02 × LAI + 0.51, with R2 and nRMSE of 0.79 and 0.13, respectively. We conclude that the N diagnostic approach developed in the present study provides a new procedure and an easy alternative tool to assess plant N status for guiding precision N management during the vegetative growth period.The accuracy of nitrogen diagnosis is vital to improve nitrogen utilization efficiency. It was shown that the standard LAI dilution curve could not be accurate across year under variable irrigation regimes. The PWC and LAI could be used to modify the standard NThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.YZ, ZL, and WY conceived, designed, and performed the experiments and analyzed the data. YZ, ZL, WY, PC, and GY discussed and drafted the manuscript. YZ, XX, JW, HF, and RC revised the manuscript and edited the English language. All authors read and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Fusarium verticillioides and Fusariumproliferatum, and the type B trichothecene deoxynivalenol, typically produced by Fusarium graminearum, are highly prevalent on cereal grains that are staples of many cultural diets and known to represent a toxic risk hazard. In Peru, corn and other cereals are frequently consumed on a daily basis under various forms, the majority of food grains being sold through traditional markets for direct consumption. Here, we surveyed mycotoxin contents of market-bought grain samples in order to assess the threat these mycotoxins might represent to Peruvian population, with a focus on corn. We found that nearly one sample of Peruvian corn out of six was contaminated with very high levels of Fumonisins, levels mostly ascribed to the presence of F. verticillioides. Extensive profiling of Peruvian corn kernels for fungal contaminants could provide elements to refine the potential risk associated with Fusarium toxins and help define adapted food safety standards.Consumption of cereals contaminated by mycotoxins poses health risks. For instance, Fumonisins B, mainly produced by  Fusarium verticillioides and Fusarium proliferatum, see Reference . In. Inchicht points . In the c health . ConsideA total of 119 cereal and pseudo-cereal samples—consisting of corn, wheat, amaranth grains , rice, barley, and oat—were collected from local Peruvian markets located in different areas of the country between 2014 and 2016 .As mentioned above, Peru can be divided into three main landscapes: the Costa , the Selva , and the Sierra that follows the Andes and contains most of the cultivated lands . Most saFusarium by qPCR detection of a conserved region of the ß-tubulin chain gene . Using an estimate of the abundance of the target by qPCR, mycotoxin risk levels related to the presence and relative abundance of Fusarium spp. were further graded “very low” , “low”, “moderate”, “high”, or “very high”. A total of 104 grain samples (85% of them) were tested positive for the presence of Fusarium spp.  . Fungal DNA abundance was estimated by external calibration using gDNA extracted from axenic cultures of Fusarium species , low , moderate (30 < Cq ≤ 35), high (25 < Cq ≤ 30), or very high (Cq ≤ 25).Analyses were performed using 20 ng of each DNA sample mixed in a 10 μL-reaction volume, using the QuantiFastcies see , which wFusarium spp. was assayed by testing for the presence of the gene encoding tubulin beta chain using broad-range primers ßT2a [Fusarium mycotoxins was estimated with the detection of genes involved in their biosynthetic pathways: Fum1 for Fumonisins, Type B and type A Tri5 alleles for type B and type A trichothecenes, respectively (see Supplementary Methods and Fusarium species that can produce the mycotoxins for which samples were found at risk was sought using species-specific primers (Fusarium species (see The presence of ers ßT2a  and ßt-2ers ßT2a  (Table 2v/v). After 1 h of agitation, 4 mL were evaporated to dryness at 50 °C under a gentle stream of nitrogen. Samples were reconstituted in 200 µL of methanol/water  before analysis. MS analysis was performed using a QTrap 2000 system  equipped with an ElectroSpray Ionization (ESI) source and an 1100 Series HPLC system . Chromatographic separation was achieved on a Kinetex XB - C18 100 Å column   protected with a guard column of the same material and maintained at 45 °C. Solvent A consisted of methanol/water  and solvent B consisted of methanol/water . Gradient elution was performed with the following conditions: 4 min with a linear gradient from 85% to 5% A, 4 min held at 5% A, 1 min linear gradient from 5% to 85% A and 85% A for 8 min post run reconditioning. The injection volume was 5 μL. The flow rate was kept at 0.7 mL/min and a split was used so that 350 µL/min was forwarded to the ESI source. The electrospray interface was used in the negative ion mode at 400 °C with the following settings: curtain gas, 20 p.s.i.; nebulizer gas, 30 p.s.i.; auxiliary gas, 70 p.s.i.; ion spray voltage, −4200 V; declustering potential, −30 V; entrance potential, −10 V. Quantification was performed using external calibration ranging from 10 to 1000 ng/mL prepared with pure standards .Ground samples were used as starting materials. The analysis of fumonisin content  was performed according to the method published by Picot and Colleagues . TCTB an


Anabolic steroids are widely administered to patients with aplastic anemia (AA) and are associated with numerous medical complications. To assist with future diagnoses, we report about a young boy with multiple hepatocellular adenomas (HAs) induced by long-term use of anabolic androgenic steroids (AAS) for AA and present a related literature review.A 15-year-old boy who was diagnosed with AA in 2011 had been treated with stanozolol (6 mg per day) and ciclosporin A (120–150 mg per day) for almost 4 years. He presented with epigastric pain and fever, and abdominal computed tomography showed a lesion of heterogenous density measuring 13.5 × 13.0 × 8.0 cm in the left hepatic lobe, which was initially misdiagnosed as a liver abscess.The patient went into hemorrhagic shock twice after invasive manipulation that aimed at diagnosis and was finally diagnosed with HA using fine needle aspiration.The patient discontinued AAS and only reserved ciclosporin A for AA treatment.Follow-up abdominal computed tomography performed 4 years after AAS discontinuation showed obvious regression of the hepatic lesions.It is of great importance for hematologists to completely understand that the long-term use of AAS may cause HA, which carries a great risk of hemorrhage and malignant transformation. Here, we report a case of AAS-induced HA who suffered hemorrhagic shock because of an invasive diagnostic operation. Our aim is to share our lessons with the wider medical field and highlight the diagnosis and treatment of AAS-induced HA.Anabolic androgenic steroids (AAS) are widely used by bodybuilders to achieve a rapid increase in muscle mass and by aplastic anemia (AA) patients to stimulate hematopoiesis. A growing number of reports suggest that the abuse of AAS is associated with serious adverse effects. Several liver disorders have been reported to be related to AAS administration, namely cholestatic jaundice, peliosis hepatis, hepatocellular adenoma (HA), and hepatocellular carcinoma.29/L (reference range: 3.5–9.5 × 109/L), a neutrophil absolute value (NE#) of 6.71 × 109/L (1.8–6.3 × 109/L), a hemoglobin (HGB) level of 61 g/L (130–175 g/L), and a platelet (PLT) count of 25 × 109/L (125–350 × 109/L). Liver function examinations were notable for elevated levels of aspartate aminotransferase (AST) at 134.0 U/L (13–35 U/L), alanine aminotransferase (ALT) at 142.7 U/L (7–40 U/L), γ-glutamyltranspeptidases (γ-GT) at 96.6 U/L (7–45 U/L), alkaline phosphatase (ALP) at 3402 U/L (50–135 U/L), total bilirubin at 55.9 umol/L (6.8–30 umol/L), and direct bilirubin at 32.0 umol/L (0–8.6 umol/L). Coagulation tests were entirely normal, as were serum levels of alpha-fetoprotein (AFP). Hepatitis virus markers, including hepatitis B and C, were negative. Computed tomography (CT) of the abdomen showed a lesion of heterogeneous density measuring 13.5 × 13.0 × 8.0 cm in the left hepatic lobe  in 2015. He was diagnosed with non-severe AA in 2011 and had been taking stanozolol (6 mg per day) and ciclosporin A (120–150 mg per day) for almost 4 years to treat the condition. Physical examination disclosed tenderness in the left upper quadrant of the abdomen, obvious hepatomegaly, and mild jaundice. A routine blood test showed a white blood cell (WBC) count of 9.69 × 10The patient was then transferred to the hematologic department to determine the exact diagnosis. Following this, a 3-phase enhanced CT scan revealed that the liver was significantly enlarged. A lesion of heterogeneous density measuring 13.5 × 13.0 × 8.0 cm was observed in the left hepatic lobe, though the margin was less clear. There were another two lesions in the left lateral hepatic lobe and the right posterior lobe, measuring 3.4 × 2.2 cm and 2.0 × 1.5 cm, respectively. Dynamic contrast study revealed no enhancement of the lesions  that range in size from a few millimeters to thirty centimeters, while AAS-induced HAs appear to predispose multiple lesions.In this case, the patient presented with epigastric pain and fever, while physical examination revealed obvious hepatomegaly and mild jaundice. During the diagnostic process of this case, we did not fully realize the hemorrhage risk of the tumor, resulting in the patient experiencing hemorrhagic shock twice; as these events were life-threatening, we must draw attention to this condition to raise the awareness of other hematologists.3.2–16 A recent study enrolled 182 individuals who used AAS for ≥6 months and found a broad spectrum of liver injuries in this cohort, including hepatotoxicity (46/182) and HA (1/182). Although AAS-induced HAs are relatively rare, the possibility that oral AAS can induce liver cell proliferation must be taken into consideration.In recent years, AAS have been proven to be involved in the development of HAs for the treatment of AA, hereditary angioedema (HAE), and muscle mass development in bodybuilding and transgender individuals.We summarized 11 recently published cases of AAS-induced HAs in Table The possibility of AAS-induced HA cannot be ruled out in our case for the following reasons. Several studies have reported stanozolol as a trigger of HA, and this patient had a 4-year history of exposure to stanozolol before the onset of HA. The clinical characteristics of this patient were consistent with those detailed in the literature; he was an adolescent male who was susceptible to developing AAS-induced HAs, and the multiple lesions seen in his liver are common indicators of AAS-induced HA. Most importantly, the lesions were significantly regressed after AAS withdrawal.3.3,19Since biochemical test abnormalities are rare in HA, multiple imaging modalities are of great importance for diagnosis, including US, CT, and magnetic resonance imaging (MRI) of the abdomen. HA is a vascular tumor with a predominantly arterial supply in classic cases. Expected findings consist of well-vascularized and well-defined solid lesions that are predominantly in an arterial phase on CT or MRI. As seen in our case, the lesions may be dyshomogeneous, especially if hemorrhage, necrosis, or fibrosis is present. The sensitivity and specificity of MRI for HA may be as high as 88% to 100%. Compared with conventional MRI, three-phase hepatobiliary MRI with delayed images has a specificity of 100% and a high sensitivity, allowing for the accurate diagnosis of HA; it is particularly valuable for HA lesions smaller than 3 cm.Dynamic MRI with a hepatocyte-specific contrast agent, such as gadobenate dimeglumine, is the best modality for diagnosing HAs. The tumor can have a clearly defined central margin with nearly parallel vessels entering from the periphery, giving the appearance of a spoked wheel. Additionally, different molecular subtypes of HA have particular B-mode echogenicity contrast-enhanced imaging appearances, and so can be complement approaches to MRI.Contrast-enhanced ultrasound (CEUS) is a rising imaging technique that is increasingly used to diagnose liver lesions, as it is important for differentiating between HA and focal nodular hyperplasia (FNH).,26 In the present case, FNA was an important procedure for confirming the HA diagnosis, but also resulted in intra-abdominal bleeding and hemorrhagic shock. Therefore, we learned that multiple HAs can be seen in AA patients who use AAS over an extended period, and FNA should not be recommended for diagnosis as life-threatening complications may occur.Percutaneous liver biopsy or FNA are usually not utilized for diagnosis because HA has a tendency to bleed following biopsy, and because the amount of tissue obtained using these methods is frequently insufficient for establishing a diagnosis.3.4Treatment of HA mainly depends on symptoms, size, number of lesions, location, and risks of hemorrhage and malignant transformation, and include cessation of associated drugs, surgical resection, transarterial embolization (TAE), thermal ablation, and liver transplantation. In case of AAS-induced HA, discontinuation of AAS may regress the mass and thereby potentially prevent unnecessary hepatic surgery. Martijn et al stated that 98% of HAs follow this trend, and a conservative approach could lead to HA regression below 50 mm. Moreover, a study of 180 patients diagnosed with HA >5 cm found that 81 patients (45%) treated with conservative therapy reached the clinical regression endpoint of <5 cm after a median of 34 months. In the cases summarized in Table Conservative management may be preferred if the HA is small (<5 cm) and associated with oral AAS. In the elective setting, TAE has also been performed as an alternative to surgical intervention. In a systematic review of 851 patients with HA, 151 patients (17.7%) underwent TAE with a reported tumor regression rate of 75%. Complete tumor disappearance was observed in 10% of patients and surgery was avoided in 45% of patients. Thermal ablation is a common minimally invasive alternative to surgery which has been recently reported as a treatment method for HA.,32 While current studies have only involved a limited number of patients, ablative methods have typically demonstrated efficacy for small HAs (<5 cm).TAE is considered as the first choice in the management of hemodynamically stable patients with bleeding HA. Therefore, HAs that do not resolve after cessation of AAS should also be considered for surgical resection. One controversial issue in the surgical management of HA is the timing of resection after cessation of AAS therapy. Liver transplantation should be reserved for patients for whom surgical resection is not possible due to tumor size/location and for those with adenomatosis.However, even after the discontinuation of AAS, growth, rupture, and malignant transformation have all been documented. Although the hepatic lesions of our patient were regressed obviously after AAS cessation, there is still a high risk of malignant transformation; therefore, a careful follow up of our patient by imaging methods and AFP every 6 months is necessary for continued management.Moreover, male gender, tumor size, and AAS-induced HA are known risk factors for malignant transformation among patients with HA.4AA patients, especially those taking AAS over an extended period, should be considered at risk of developing HA. Multiple imaging modalities are considered to be the most important methods for diagnosis, while FNA should be used with caution as life-threatening bleeding complications may occur. If HA is diagnosed, or even suspected, the use of AAS should be discontinued as soon as possible and the appropriate treatment strategy should be carefully selected according to the individual's condition. Because of the high risk for malignant transformation, patients should also be monitored carefully with biochemical analyses of liver function, AFP serum levels, and US studies.We are grateful to the patient and his family, who gave his informed consent for publication.Investigation: Yuying Li,Hongqiong Fan, Yanhua Zhou,. Gnana Bharathi.Radiologic imagines: Fanyang Meng.Resources: Wei Li.Writing – original draft: Lixia Wang, Cong Wang.Writing – review & editing: Sujun Gao, Yan Yang.


The light response curve indicated that when vanilla was exposed to 1000 μmol m−2s−1, the electron transport rate (ETR) and photochemical quenching of fluorescence (qP) were significantly reduced to a negligible amount. We found that if a vanilla orchid was irradiated with BL100 for 12 days, it acquired BL-acclimation. Chloroplasts moved to the side of cells in order to reduce light-harvesting antenna size, and chloroplast photodamage was eliminated. Therefore, BL-acclimation enhanced vanilla orchid growth and tolerance to moderate (500 μmol m−2s−1) and high light (1000 μmol m−2s−1) stress conditions. It was found that under high irradiation, BL-acclimatized vanilla maintained higher ETR and qP capacity than the control without BL-acclimation. BL-acclimation induced antioxidant enzyme activities, reduced ROS accumulation, and accumulated more carbohydrates. Moreover, BL-acclimatized orchids upregulated photosystem-II-associated marker genes (D1 and PetC), Rubisco and PEPC transcripts and sustained expression levels thereof, and also maximized the photosynthesis rate. Consequently, BL-acclimatized orchids had higher biomass. In short, this study found that acclimating vanilla orchid with BL before transplantation to the field might eliminate photoinhibition and enhance vanilla growth and production.Vanilla orchid, which is well-known for its flavor and fragrance, is cultivated in tropical and subtropical regions. This shade-loving plant is very sensitive to high irradiance. In this study, we show that vanilla chloroplasts started to have avoidance movement when blue light (BL) was higher than 20 μmol m Orchidaceae) and boast the family’s only edible fruit. Vanilla orchids are distributed between 27 °N and 27 °S on all continents [Vanilla planifolia, the most popular cultivated species of vanilla, known as “Bourbon” or “Mexican” vanilla, is native to the tropical forests of Central America and Mexico and was historically cultivated by the peoples of Mesoamerica. Vanilla pods produce vanillin, which is a fragrant compound with high economic value. Today, market demand for natural vanillin is increasing, and vanilla orchid is cultivated in many tropical and subtropical zones [2 absorption during the night (Phase I), thus affecting the abundance of carbohydrate production through the Calvin cycle. Vanilla orchid is a shade-loving plant that is very sensitive to high radiation. For vanilla orchid production, support trees should have a dense canopy to provide 70% to 80% shade [2 uptake. It has also been found that vanilla has higher photosynthesis and biomass growth under intermediate levels of radiation of 17% to 31% relative illumination [Vanilla orchids make up about 110 species in the orchid family ) and light-independent  pathways. Light reactions take place in the chloroplast thylakoid membrane for the splitting of two water molecules into one oxygen molecule and four protons and electrons. Then, the protons and electrons are transferred through the thylakoid membrane to create the energy-carrying molecules adenosine triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH). ATP and NADPH are then used by RuBP carboxylase/oxygenase for the Calvin cycle to convert COhydrates . PSII anhydrates . Excess hydrates .3 plants [−2s−1, chloroplasts start to show avoidance movement and significant chloroplast avoidance has been found in the strong blue light of 100 μmol m−2s−1 [Arabidopsis, tobacco and rice increased photosynthesis efficiency and significantly enhanced crop yield [Arabidopsis growth and seed yield [Setaria viridis [To prevent photoinhibition and to acquire acclimation to light-fluctuating environments, plants have developed direct and indirect mechanisms for sensing and reacting to the high light irradiation . Photopr3 plants . In CAM l m−2s−1 . When exl m−2s−1 ,13. Planl m−2s−1 . D1 , such as singlet oxygen and Hss light . Plants ss light . Moreovess light .To date, basic studies on vanilla orchid are scarce. In our previous study, we showed that blue light mediated the photoacclimation of moth orchid . We, the−2s−1 for 5 h. Slit assay results indicated that BL-treated leaves have a light green color, but RL-treated leaves do not  data were recorded every hour. ANOVA analysis data indicated that BL intensity significantly affected the chloroplast movement of L3 and LM, but the interaction of BL × Time was of no significant difference (phot1 and phot2. qRT-PCR results indicated that young leaves (L3) expressed a similar amount of phot1 and phot2 at BL5, and phot2 was expressed at a higher level than phot1 in the dark and under BL100 , with various light intensities at 100, 150, 200, 300, 400, 500, and 1000 μmol muced ETR a. NPQ shuced ETR b. The qPl m−2s−1 c.−2s−1 and set the temperature to 21, 24, 26, 30, 33, or 36 °C and detected Chl F parameters. Results indicated that 21 °C had the lowest ETR and qP but increased when the temperature increased. Vanilla orchid tended to have the highest ETR and qP at 33 °C, then gradually decreased again when the temperature increased up to 36 °C  for 0, 4, 8, or 12 days. In order to investigate the effect of light intensity on the Chl F capacity of vanilla orchid, we set the fluorescence leaf chamber with light (composed of 90% red light and 10% blue light) intensities of 150, 500, and 1000 μmol m−2s−1, respectively, and detected the Chl F parameters. Analysis of variance (ANOVA) statistical analysis indicated that all the Chl F parameters had a significant difference between the various BL-acclimation time courses. It is found that light intensities were significantly different for ETR, NPQ, qP, and qN parameters. There was also a significant difference in BLday × light interaction for the parameters of ETR and qP  (LHCA1), PSII, and PSI, but significantly upregulated transcript levels of D1 and PetC (Rubisco (RBCL2) and PEPC were also upregulated after BL-acclimation for 12 days (BL-acclimation of vanilla orchid significantly upregulated transcript levels of blue-light-responsive genes such as  (ELIP1) a. BL-accand PetC b. Transc 12 days c.p < 5%) between those with or without BL-acclimation. However, when exposed to HL1000, the vanilla orchids that had been BL-acclimated sustained significantly higher Chl F capacity for Fv/Fm, ETR, NPQ, qP, and qN than the control without BL-acclimation  and high irradiation (HL1000), respectively. After high irradiation for two weeks, vanilla orchid with BL-acclimation had better growth and biomass  and highlimation . ClearlyVanilla orchids were treated with or without BL-acclimation then exposed to ML500 and HL1000 irradiation, respectively. After irradiation for two weeks, L3 leaves were collected and SOD and CAT enzyme activities were detected. Glucose and sucrose content were also compared in the control and BL-acclimated vanilla orchids. Results indicated that BL-acclimation significantly increased antioxidant enzyme activity of SOD after exposure to ML500 or HL1000 a and CATphot1 mRNA at ML500  and PEPC were expressed at higher levels in those vanilla leaves pretreated with BL-acclimation than the control without BL-acclimation  [−2s−1, the young and mature leaves both started to exhibit chloroplast avoidance to reduce photoinhibition. Obvious chloroplast avoidance movement of vanilla was found when it was exposed to high BL irradiation of 100 μmol m−2s−1   and othe orchid) ,24. A SPl m−2s−1 c,d. Youne leaves e,f, implh orchid . Although orchid .−2s−1) but started decreasing when light is higher than 150 μmol m−2s−1. When vanilla was exposed to light higher than 300 μmol m−2s−1, qP significantly reduced to one-third of that at the irradiation of 100 μmol m−2s−1. Vanilla significantly lost ETR and qP when exposed to the high light of 1000 μmol m−2s−1  and temperature (33 °C) that may enhance vanilla photosynthesis efficiency and growth. However, under field plantation, the temperature is usually higher than 36 °C, and high irradiation fluctuates; hence, incorporating high temperature and high irradiation tolerant traits together in a vanilla orchid breeding program may boost crop production.The light response curve of Chl F parameters showed that vanilla orchid young leaf has the highest photochemical quenching of fluorescence (qP) under the low light condition  [phot1 and phot2 transcripts and mediated chloroplast avoidance movement; thus, chloroplasts moved to the side of cells  . Moreove orchid) ,29. Thisof cells c, causinof cells f. A reduAnthocyanin pigments reduce the photoinhibition and photobleaching of chlorophyll under high light stress conditions . However2O2 in Arabidopsis plasma membrane and chloroplasts and regulated chloroplast avoidance [SOD and CAT mRNAs and turned on antioxidative enzyme activities to carry out the ROS scavenging process to detoxify ROS , a component of Cyt b6f complex, induces D1 and increases the photosynthesis rate and plant biomass [D1 and PetC are upregulated during BL-acclimation as well as after high irradiation exposure  with five to six leaves were purchased from Vanilla Knight Co.  and planted in the AS-BCST greenhouse with 50% shading, at a temperature of 26 ± 3 °C. In order to know the light and temperature requirement for vanilla orchid, we carried out a light response experiment  and studied the effect of temperatures  on Chl F parameters of vanilla. Each treatment was conducted on three plants.Potted vanilla orchid plants  under LED white light at 500 μmol m−2s−1  or high irradiation of 1000 μmol m−2s−1 . After high irradiation for two weeks, chlorophyll fluorescence was measured in three plants, and three plants were collected for RNA, ROS, and biochemical analysis. Growth chambers were set at a constant 26 °C, 12 h light/12 h dark period, whilst the other set of vanilla plants were kept in white light without BL-acclimation and then exposed together with the BL-acclimated plants to ML500 and HL1000, respectively.To identify how many days is sufficient to acquire BL-acclimation in vanilla orchid, we treated vanilla plants with BL100 in a constant 26 °C growth chamber for 4, 8, and 12 days, and the control was without BL-acclimation. Each treatment was conducted on three plants. To further study the effect of BL-acclimation protection on vanilla orchid exposure to high irradiation, vanilla plants were separated into two parts (control and BL-acclimation). A total of 18 plants were BL-acclimatized in a growth chamber set at a constant 26 °C and continuous irradiation with light-emitting diode (LED) blue light  at 100 μmol m−2s−1, respectively, and SPAD data of the L3 leave were measured every hour for a total of five hours. SPAD data were normalized to the initial data after dark-adaptation.To assay nondestructive chlorophyll content, we used a soil plant analysis development (SPAD) chlorophyll meter (model SPAD-502). To detect the chloroplast movement of vanilla, vanilla plants were dark-adapted overnight, and the new fully expanded leaf  and mature leaf (LM) were used to measure SPAD, following the procedures  and prot2 leaf area aperture for the Chl F measurements. Leaf chamber temperature was set at 26 °C, and CO2 concentration was set at 400 ppm. L3 leaves were covered with aluminum foil and dark-adapted more than 2 h before measuring the parameters of chlorophyll fluorescence. A saturating actinic light pulse of 8000 μmol m−2s−1 for one second was applied to measure the maximal fluorescence. Actinic light is composed of 90% red light (625 nm) and 10% blue light (470 nm). Data of Fv/Fm, electron transport rate (ETR), nonphotochemical quenching (NPQ), photochemical quenching of fluorescence (qP), and nonphotochemical quenching of fluorescence (qN) were recorded and analyzed. Each treatment has three bioreplicate measurements.Chlorophyll fluorescence (Chl F) parameters were measured using a LiCOR 6800 system  equipped with a fluorescence leaf chamber 6800-01A  and 2 cmVanilla orchid newly expanded L3 leaf samples, with or without BL-acclimation for 12 days, were exposed to ML500 or HL1000 for two weeks and then collected and stored at −80 °C freezer. A total of 100 mg of frozen leaf samples were homogenized. SOD enzyme activity was determined using a Superoxide Dismutase (SOD) Activity Colorimetric Assay Kit (BioVision #K335-100), following the instruction of the manufacturer. SOD enzyme activity was calculated based on the percentage inhibition after 20 min of incubation at 37 °C. For detection of catalase (CAT) enzyme activity, 100 mg frozen leaf samples were homogenized and determined using a Catalase Activity Colorimetric/ Fluorometric Assay Kit , as described previously .Vanilla orchid L3 leaves, with or without BL-acclimation for 12 days, which had then been exposed to moderate light (ML500) or high light (HL1000) irradiation for two weeks, were collected. A total of 100 mg of leaf samples were homogenized, 500 μL of 1 × PBS buffer was added, and then the mixture was incubated for 30 min at 37 °C. Then, glucose and sucrose concentrations were determined using a Glucose and Sucrose Colorimetric/Fluorometric Assay Kit (Sigma #MAK013), following the instructions provided by the manufacturer.Ubiquitin (VPTC006466), as a control.L3 leaf samples from vanilla orchids, with or without BL-acclimation for 12 days, which had then been exposed to ML500 or HL1000 for two weeks, were collected. Total RNA was isolated and DNase treated, then 1 μg RNA was reverse-transcribed to 1st cDNA using the M-MLV reverse transcriptase cDNA synthesis kit , and then qRT-PCR was performed for 40 cycles with KAPA SYBR FAST qPCR Master Mix , as described previously [p-values of less than 0.05 were considered statistically significant. Dunnett’s test was used to compare the difference between BL-acclimation and the control without BL-acclimation.Statistical analyses were performed using Statistical Analysis System (SAS) software version 9.4, using the analysis of variance (ANOVA) procedure, followed by Duncan’s multiple range test. −2s−1 for 12 days induces photoacclimation in this orchid species. Blue light triggers biochemical and physiological processes that confer better recovery when vanilla orchids are exposed to high irradiation. Vanilla blue-light photoreceptors, phototropins, sense the high fluence rate of blue light and upregulate phot1 and phot2 gene expression and mediate chloroplast avoidance in order to reduce photodamage. BL-acclimation enhances antioxidant activities and decreases ROS toxicity in vanilla orchid, resulting in increases in the transcript levels of PSII, D1, and PetC, enhancing PSII repair and sustaining the photosynthesis rate under conditions of high irradiation. Hence, acclimating vanilla orchids with BL before transplantation to the field might enhance photoprotection and improve its growth and production. In the future, the knowledge gained from this study may help to manipulate the photosynthesis efficiency of vanilla orchid and improve its resilience to environmental stress. In addition, BL-acclimation may be applied to other crops to hardening seedlings, increase field survival, and increase crop yields.Collectively, this study demonstrates that pretreated vanilla under strong blue light at 100 μmol m