Integrative transcriptome and proteome analysis identifies major

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Integrative transcriptome and proteome analysis identifies major metabolic pathways involved in pepper fruit development Zhoubin Liu, Junheng Lv, zhuqing Zhang, Heng Li, bozhi Yang, wenchao chen, xiongze Dai, xuefeng Li, Sha Yang, li Liu, lijun Ou, yanqing Ma, and Xuexiao Zou J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00673 • Publication Date (Web): 17 Jan 2019 Downloaded from http://pubs.acs.org on January 17, 2019

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Journal of Proteome Research

Integrative transcriptome and proteome analysis identifies major metabolic pathways involved in pepper fruit development Zhoubin Liu1,2#, Junheng Lv1,2#, Zhuqing Zhang2#，Heng Li3, Bozhi Yang2, Wenchao Chen2, Xiongze Dai2, Xuefeng Li2, Sha Yang2, Li Liu3, Lijun Ou2*, Yanqing Ma2*, Xuexiao Zou1,2*

1 Longping Branch, Graduate School of Hunan university, Changsha, 410125, China 2 Vegetable Institution of Hunan Academy of Agricultural Science, Changsha, 410125, China 3 Shanghai Applied Protein Technology Co. Ltd, Shanghai, 200233, PR China

# These authors contributed equally. *Corresponding author： Lijun Ou: [email protected]. Yanqing Ma: [email protected]. Xuexiao Zou: [email protected]. Tel: 86 0731 84692619, Fax: 86 0731 84692619

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ACS Paragon Plus Environment

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Abstract Pepper (Capsicum annuum L.) fruit development is a complex and genetically programmed process. In this study, we conducted integrative analysis of transcriptome and proteome profiles during pepper fruit development. A total of 23,349 transcripts and 5,455 protein groups were identified in four fruit developmental stages of two pepper varieties. The numbers of transcripts and proteins identified were decreased gradually across fruit development and the most significant changes in transcript and protein levels happened from the mature green (MG) to breaker (Br) stages. Poor correlation between differentially expressed transcript and differentially expressed protein profiles was observed during pepper fruit development. We then analyzed expression profiles of transcripts and proteins involved in cell wall metabolism, and capsanthin, tocopherol and ascorbate biosynthetic pathways during fruit development, and identified key regulatory proteins in these pathways. We presented a dynamic picture of pepper fruit development via comprehensive analysis of transcriptome and proteome profiles at different fruit developmental stages and in different varieties, revealing the temporal specificity of key protein expression. Our report provides insight into the transcription and translation dynamics of pepper fruit development and a reference for other non-climacteric species.

Key words: pepper; fruit development; transcriptome; proteome; cell wall; capsanthin; tocopherol; ascorbate.

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Introduction Pepper (Capsicum annuum L.) is one of the most important vegetable crops worldwide. Besides widely used as vegetables and spicy ingredients, pepper is also used as medicines, cosmetics, natural coloring agents and ornaments1, 2. Pepper fruits have significant diversity in morphology, nutrition, and color. Fruit ripening in pepper is not accompanied by a large amount of ethylene synthesis, thus providing a good model for the developmental biology of non-climacteric fruits3. Recently, researchers have studied capsaicin4, antioxidant5, RNS6 and mineral element absorption to explore the developmental physiology of pepper fruit7. However, these studies cannot provide a global picture of regulatory networks of the pepper fruit development, as well as the relationship between genetic and metabolic regulation during fruit development and ripening. In recent years, to better understand fruit development and ripening mechanisms, numerous studies have focused on transcriptome profiles using RNA sequencing (RNA-Seq) technology, e.g., in tomato8, sweet orange9, cherry10 and peach11. In pepper, Curry et al.12 identified three specific enzyme genes participated in capsaicin biosynthesis by screening a pepper (C. chinense) placenta cDNA library and the transcript levels of these three genes were positively correlated with capsaicin production. Martínez-López et al.13 investigated transcriptomes of pepper fruits at different developmental stages, and found that genes related to the synthesis of capsaicin and vitamin C were significantly up-regulated at immature green stage. Recently, Liu et al.14 generated a large-scale transcriptome profile dataset for an elite pepper breeding line through high throughput mRNA sequencing and established a public data platform ‘pepperHub’ for pepper research. Transcriptional levels are only a moderate predictor for protein expression as they do not account for posttranscriptional processes such as translational regulation or protein stability. Although substantial progress has been made on understanding the transcriptome dynamics of fruit development, the dynamic changes or biochemical regulation of proteins during fruit development still remain largely unexplored15. Proteins are thought to have a more direct correlation with metabolites, compared to mRNAs16. Nowadays, proteomics has been 3



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successfully applied in strawberries17, pear18, citrus19, 20, tomatoes21 to investigate the changes of protein components during fruit development. Prinsi et al.22 identified 53 differentially expressed proteins during peach fruit ripening using the 2-DE approach, and most of these proteins were involved in ethylene synthesis, stress responses and primary metabolisms. Nogueira et al.23 identified 37 significantly different proteins in papaya fruits before and after respiratory jump. The proteomics researches on pepper have been conducted on those related to subcellular function24, disease resistance25 and cytoplasmic male sterility26,

27

. Lee et al.28 performed

comparative proteome analysis of placental tissues between pungent and non-pungent pepper cultivars and identified candidate proteins involved in the regulation of pepper pungency. However, to the best of our knowledge, no large-scale proteomic analyses related to pepper fruit developmental process have been reported. Previous studies have shown that the combination of proteome and multiple histology can be used to study the physiological and biochemical changes during fruit ripening29. Combined transcriptome and proteome analyses have been conducted to obtain better understanding of various biological processes such as circadian clock, chloroplast development and disease responses30,31,32. However, similar research on fruit development has been limited to a few crops including mango33, citrus34 and Litchi35. In this study, we used RNA-Seq and label-free quantitative (LFQ) proteomic approaches to comprehensively investigate the global transcriptome and proteome profiles of two pepper varieties to gain a broader systematic view of pepper fruit development and ripening, and to identify additional common as well as distinct molecular regulatory events at different developmental and ripening stages. This study provides a detailed framework of fruit pericarp proteome dynamics, and the association and difference with transcriptome profiles at different stages of pepper fruit development and ripening.

Materials and Methods Plant growth and sampling Two pepper (Capsicum annuum L.) varieties, ‘SJ11-3’ and ‘06g19-1-1-1’, were provided by 4


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Hunan Vegetable Research Institute (Changsha, China), and grown in a greenhouse (16 h of light at 30±2 ℃ and 8 h of darkness at 20±2 ℃). Fruits at the following developmental stages, immature green (IMG; 20 days after anthesis (DAA)), mature green (MG; 30 DAA), breaker (Br; 40 DAA) and mature red (MR; 50 DAA), were collected for physiological, transcriptome and proteome analyses (Figure 1). Fruit pericarps at each developmental stage were collected and pooled from three individual plants. The fruits pericarp tissue was then divided into three parts, two frozen in liquid nitrogen, stored at -80°C for transcriptome and proteome analyses, and one used for physiological measurement. Three biological replicates in each time point were performed.

Figure 1 Measurement of ascorbic acid and tocopherol contents Total ascorbic acid content of pepper fruit samples was determined according to the method described by Zhang et al.36. Total tocopherol content was determined as described by Baydar and Erbaş 37. Three biological replicates were analyzed for each sample. Data were analyzed with SPSS v22.0. Significance of differences among different samples was determined using Duncan's multiple comparison test. Measurement of capsanthin The capsanthin were incubated at 60 ºC to completely dry, and then ground into paprika, and 5



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used for capsanthin extraction with 100 mL acetone. The extraction solution was filtered, and diluted ten times with acetone before colorimetric analysis. The absorbance at 460 nm of the diluent (about 2 mL) were read by a UV-1780 spectrophotometry (Shimadzu, Japan), and pure acetone

was

served

as

the

blank

control.

The

color

value

was

calculated

as

E_460nm^(1%)=AF/M, where A is the absorbance value at 460 nm of a sample using 1 cM cell, F is the dilution factor, and M is the dry mass of a sample. Transcriptome analyses Methods S-1 provides details on RNA extraction, RNA-Seq library preparation and sequencing, and bioinformatics analyses2,38,39,40,41,42. Proteome Analyses Methods S-2 provides details on sample preparation, SDS-PAGE separation and FASP digestion, HPLC and LC-MS/MS analyses, gene ontology annotation, KEGG pathway annotation, and functional enrichment analysis (Figure S-1)43,44,45,46,47,48. Correlation analyses of transcriptome and proteome profiles A gene and its corresponding protein are considered to be correlated in compared stages if both the gene and protein were expressed at these stages. Next, the significance of the expression between the compared stages for both correlated transcripts and proteins was determined. If both a gene and its corresponding protein both showed significant difference in their expression levels between the compared stages, they were defined as differentially expressed correlated transcript (DECT) and protein (DECP), respectively (Table S-1). Gene ontology (GO) term annotation, KEGG pathway annotation, and functional enrichment analysis were then conducted for DECTs and DECPs, and the Pearson correlation coefficient was calculated49 for each pair of DECT and DECP in pepper fruits during development. Quantitative real time PCR qRT-PCR was conducted as described by Osorio et al.50. Three replicates were performed for each sample. CpActin gene was used as the internal control. Primers used in the study are listed in Table S-2. The relative gene expression levels were normalized using the 2-ΔΔCT method51. Targeted protein quantification by parallel reaction monitoring (PRM) 6


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PRM analyses were performed as described by Kim et al.52. Peptide sequences used for PRM were listed in Table S-3. Each sample was analyzed separately by LC-PRM/MS. Briefly, peptides were prepared according to the label free protocol, and a PRTC stable isotope peptide was spiked in each sample as internal standard reference. Tryptic peptides were loaded onto the tip of the C18 stage for desalination prior to reversed-phase chromatography on an Easy nLC-1200 system (Thermo Scientific). One hour liquid chromatography gradients with acetonitrile ranging from 5 to 35% in 45 min were used. PRM analysis was performed on a Q Exactive HF mass spectrometer (Thermo Scientific). A unique peptide with high strength and confidence for each target protein was used to optimize the collision energy charge state and retention time of the most significantly regulated peptide through methods. The mass spectrometer was operated in positive ion mode and with the following parameters: The full MS1 scan was acquired with the resolution of 60000 (at 200 m/z), automatic gain control (ACG) target values 3.0×10-6, and a 200 ms maximum ion injection times. Full MS scans were followed by 20 PRM scans at 35000 resolution (at m/z 200) with AGC 3.0×10-6 and maximum injection time

120ms.

The

targeted

peptides

were

isolated

with

a

1.6Th

window.

Ion

activation/dissociation was performed at normalized collision energy of 27 in a higher energy dissociation (HCD) collision cell. The raw data were analyzed using Skyline (MacCoss Lab, University of Washington) where signal intensities for individual peptide sequences for each of the significantly altered proteins were quantified relative to each sample and normalized to standard reference. Accession numbers RNA-Seq data generated in this study is available from the Sequence Read Archive (SRA; http://www.ncbi.nlm.nih.gov/sra) under accession number: PRJNA485468. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (https://www.ebi.ac.uk/pride/archive/) via the PRIDE partner repository with the dataset identifier PXD010746. The mass spectrometry proteomics data of PRM have been deposited to the ProteomeXchange 7



Consortium(https://www.ebi.ac.uk/pride/archive/) via the PRIDE partner repository with the dataset identifier PXD010755.

Results and Discussion Specificity of transcript and protein accumulation at different developmental stages of pepper fruit The two selected pepper varieties display substantial differences in fruit morphology and quality. Fruit of ‘SJ11-3’ is long and screw-shaped, with better flavor and high vitamin C content, and is widely used for fresh eating, while fruit of ‘06g19-1-1-1’ is short, with poor taste quality but high pigment content, and is mainly used for the extraction of capsanthin. High throughput RNA sequencing of fruit samples at four different developmental stages in the two varieties, each sample with three biological replicates, resulted in a total of 1,454,471,486 cleaned high-quality reads (corresponding to 129.29 Gb transcriptome data), after removing low quality and adaptor sequences. The percentage of Q20 and Q30 bases in the raw data were 96.4%-98.4% and 92.0%-96.0%, respectively. High correlations were observed between biological replicates (Fig. S2). All these results indicated the high quality of the transcriptome sequencing data. After mapping the reads to the C. annuum L_Zunla-1 reference genome, 23,349 of the 35,336 genes predicted from the genome were found to be expressed in at least one sample (Table S-4). Among these genes, 17,361 and 17,083 were found to be expressed at all stages in SJ11-3 and 06g19-1-1-1, respectively (Figure 2-A). Our proteomics analysis identified 34,617 (89.79％) unique peptides from a total of 38,551 detected peptides (FDR≤0.01) (Table S-5). A total of 5,455 protein groups (FDR≤0.01) were identified based on these peptides, and 4,581 protein groups were found to contain at least two unique peptides (Table S-6). Proteome coverage was highly reproducible across the biological replicates; 80.2％-84.7％ of all identified proteins were reproducible in all three biological replicates, and 15.4%-19.8% of identified proteins were reproducible in two biological replicates. Proteins identified only in one biological replicate were excluded from downstream analysis. 8


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Among these proteins, 2,568 of 4,335 identified proteins in SJ11-3 were found in all four stages, and 2,139 of 4,154 identified proteins in 06g19-1-1-1 were found in all four stages (Figure 2-B). The numbers of proteins identified in the IMG and MG stages were similar, but much less proteins were identified in the Br stage in both pepper varieties.

Figure 2 Principal component analysis (PCA) were performed on both transcriptome and proteome profile data to visualize biological variability of fruit developmental process. The results indicated that the dominating source of variance was the differential protein/transcript signal in the two pepper varieties across all developmental stages (PC1), and the variance described by PC2 was related to different developmental changes in these two pepper varieties (Figure 3).

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Figure 3 Correlation of transcript and protein profiles during pepper fruit development In comparisons of two consecutive developmental stages of the same variety, different from the number of expressed transcripts, the number of detected proteins in SJ11-3 was always higher than that in 06g19-1-1-1 (Table 1). Although MG/IMG fruits had the largest number of detected transcripts and proteins, this comparison had the fewest differentially expressed proteins or transcripts (Table 1, Table S-7 and Table S-8). Through correlation analyses, we found that although most proteins were correlated with their corresponding transcripts, but the correlation between DEPs and DETs was very poor. In MG/IMG, Br/MG and MR/Br comparisons, only 22.1%, 66.7% and 26.1% DEPs correlated with DETs in SJ11-3, respectively, only 20.2%, 38.5% and 18.2 DEPs correlated with DETs in 06g19-1-1-1, respectively (Figure S-3). Overall, the Br/MG comparison not only had the largest number of differentially expressed transcripts (DETs) and differentially expressed transcripts (DEPs), but also had the most correlated differentially expressed transcripts and proteins, in both pepper varieties. The transition from the mature green stage to the breaker stage (color transition) of pepper fruit displayed the most significant changes in gene and protein expression. These changes not only reflected in the total number of identified transcripts and proteins, but also in the number of differentially expressed proteins, indicating that the regulatory network during this transition of pepper fruit development was more complicated than other developmental stage transitions. 10


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GO term analysis of differentially expressed correlated proteins Gene ontology (GO) term database was used to categorize the identified differentially expressed correlated proteins (DECPs). These proteins covered a wide range of biological processes, molecular functions, and cellular components. We selected the top 20 most abundant GO terms for detailed analysis (Figure 4). In terms of the number of correlated proteins, the largest group within biological process was metabolic process, followed by cellular process and response to stimulus. Catalytic activity and binding were predominant in the category of molecular function. The cellular components of these proteins mainly included cell, cell part, organelle and membrane. All these GO terms maintained the largest DECP numbers and the proportion was stable in all the three comparisons between consecutive developmental stages.

Figure 4 In the three comparison groups, although Br/MG fruit depicted the most DECPs involved in different function categories, the numbers of DECPs belonging these top 20 categories were relatively close between the two pepper varieties. The MR/Br and MG/IMG had less DECPs than Br/MG but there were the largest differences in GO term categories especially in MG/IMG fruits between the two varieties. These differences were mainly reflected in the metabolic 11



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process of biological process, the catalytic activity and binding of molecular function, and the cell, organelle and cell part of cellular component. This indicated that these functional categories might play an important role in pepper fruit development and ripening.

Expression of fruit enlargement and ripening related genes and proteins In fruits of many plant species, especially tomato, studies have been carried out to identify genes and proteins that control fruit enlargement and ripening, yet little of them was reported in pepper. In this study, based on integrated transcriptome and proteome analysis, we identified 45 fruit

enlargement-

and

ripening-related

transcripts/proteins

which

were

significantly

differentially expressed between two varieties and/or between different developmental stages, to examine the transcript-protein correlations (Figure 5). An obvious correlation between transcript and protein profiles was observed; for example, profiles of 20 proteins displayed high correlations (Pearson’s r > 0.7) with profiles of their corresponding transcripts, and 19 of these 20 proteins showed significant correlations (P < 0.05); 13 proteins displayed moderate correlation (0.4 < r < 0.7) with transcripts and only 4 showed negative correlation. Overall, these proteins showed a relatively high correlation with their transcripts during pepper fruit development. At the same time, we also found that there was a high correlation between protein expression during fruit development between the two pepper varieties, with 26 proteins having r > 0.7 and 15 of them being significantly correlated (P < 0.05).

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Figure 5 The enlargement of the pepper fruit is basically completed before its color transition. In the early developmental stages, the fruit growth is mainly the result of cell division, and the fruit continues to grow through cell expansion. Many of the 45 proteins described above play a key role in cell wall metabolism and enlargement of pepper fruit. During plant growth, the increase in cell number and volume is accompanied by changes in xyloglucan content and cell wall remodeling, while plant cell wall is a complex network structure containing cellulose, hemicellulose, and pectin and a small amount of structural proteins53. As the most important hemicellulose

in

the

cell

wall,

the

ratio

of

xyloglucan

to

xyloglucan

endotransglucosylase/hydrolase (XTH) determines the elongation rate of the cells. When the ratio of XTH to xyloglucan is high, XTH will enter the cell wall as a free enzyme and directly cut and re-cut xyloglucan, thereby relaxing the cell wall and promoting cell elongation54. In this study, XTH1 (Capana08g001512), XTH9 (Capana07g001548) and XTH32 (Capana09g002242) of the XTH family were highly expressed in the IMG and MG stages, while their abundance in the Br and MR stages was reduced, consistent with the expansion of the pepper fruit. In the development of tomato and cherry fruits, it was found that XTH may also be involved in the cell division and expansion of green fruits and the cell softening process in the mature stage, and its 13



activity in the green fruit strongly influences the final size of the fruit55, 56. This indicates that the different genes in XTH family have different functions in cell division and cell enlargement in different crops. In pepper, XTH1, XTH9 and XTH32 play important roles in original cell wall repair and reconstruction of dividing cells in fruit during expansion. Cellulose is synthesized by cellulose synthases located in the plasma membrane. Decreased cellulose synthesis will affect the growth of pollen tubes, leading to loose pollen wall and pollen tube bending57. Mutation of KOB1, a gene involved in cellulose synthesis, affects the synthesis of cellulose in Arabidopsis root cells, making the plants short58. The high abundance of cellulose synthase CSLA2 (Capana06g000581, Capana11g000211) and CSLA9 (Capana11g000211, Capana10g001944) at IMG and MG stages in pepper fruit could contribute to the synthesis of cellulose during fruit expansion, and provide more essential cell wall components for the increase of cell number and volume. Studies have shown that fruit softening is mainly the result of the interaction between polygalacturonase (PG) and pectin methylesterase (PME). As the activity of these two enzymes increases, the pectin material decomposes and the fruit hardness decreases59. In this study, the abundance of PG was too low to be detected before the Br stage, while PME was highly expressed at the IMG and MG stages. Without the action of PG in the stage of fruit enlargement, PME protein promotes the conversion of pectin in the cell wall structure into pectic acid, which promotes the relaxation of cell wall, leading to the cell growth. At the same time, we found that among the highly expressed proteins related to fruit enlargement, expression of PME2.2 (Capana00g004152) and ANN4 (capana08g001266) in fruit was significantly higher in SJ11-3 than in 06g19-1-1-1. Previous studies have suggested that the annexin protein could be involved in exocytosis of cell wall degrading enzymes, acting to sequester Ca2+ ions released from the degrading cell wall matrix, and it also play a role in regulating cell expansion during fruit development60,61,62. It may suggest that PME2.2 and ANN4 proteins have important regulatory effects on the final formation of pepper fruit size.

Dynamic changes of correlated protein abundance at fruit color-changing stages 14


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In order to study whether there is a significant change (P≤0.05) of protein abundance in the metabolic pathways (top 30), a statistical analysis was performed on the correlation data at three key fruit color-changing stages (i.e. MG, Br and MR). The top 30 metabolic pathways with the most protein abundance changes were identified. The dynamic changes of protein abundance from MG to MR could be classified into 9 patterns: stable, continuous increase, early increase, increase-decrease, late increase, continuous decrease, early decrease, decrease-increase and late decrease (Figure 6 and Table S-9). A total of 333 correlated proteins in these top 30 pathways were identified, and they display different patterns based on this classification in the two varieties (if one protein had different expression patterns in the two varieties, it was counted twice): 96 were stable, 93 underwent an increase (15 continuous + 52 early + 26 late), and 122 underwent a decrease (35 continuous + 60 early + 27 late). Among the 237 (approximately 71.2% of the 333 proteins examined) correlated proteins whose abundance was changed, 184 showed changes from MG to Br stage, with 86 increased (19 increase-decrease + 52 early + 15 continuous) and 98 decreased (3 decrease-increase + 60 early +35 continuous), and 134 (54.7%) showed changes from Br to MR stage, with 44 increased (3 decrease-increase +26 late +15 continuous) and 81 decreased (27 late + 35 continuous +19 increase-decrease) (Figure 6-A).

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Figure 6 We further studied different metabolic pathways that protein abundance had changed at three key color-changing stages (Figure 6-B). A total of 16 (9 increase-decrease + 7 early increase) of 20 proteins and 14 (7 increase-decrease + 7 early increase) of 25 proteins identified in the endoplasmic reticulum pathway in SJ11-3 and 06g19-1-1-1, respectively, showed highly increased abundances from MG to Br stage, while 10 and 17 proteins had highly decreased abundances from Br to MR stage in SJ11-3 and 06g19-1-1-1, respectively. This indicated that the processing procedure of endoplasmic reticulum was more active before the Br stage. Six (4 early increase +2 late increase) and eight (7 early increase +1 late increase) proteins involved in the fatty acid degradation pathway showed increased abundances from MG to MR in SJ11-3 and 06g19-1-1-1, respectively. In contrast, proteins in a number of metabolic pathways showed decreased abundances during fruit development. For example, proteins in the photosynthesis pathway showed decreases in abundance, with 9, 7 and 7 proteins having a continuous, an early and a late decrease in SJ11-3, respectively, and 14, 5 and 2 proteins having a continuous, an early and a late decrease in 06g19-1-1-1, respectively. The majority of the proteins in the photosynthesis-antenna proteins pathway also showed the most remarkable decrease in abundance. These two metabolic pathways were closely related to photosynthesis and energy metabolism in plants. The decrease in the abundance of genes and proteins associated with photosynthesis during fruit development has been reported before63. At the early stage of pepper fruit, high abundance of proteins in photosynthesis-related pathway provides energy for fruit expansion and development. With the complete of fruit expansion and the fruit into the mature stage, the energy required for its growth is reduced, leading to a decrease in the expression of photosynthesis and photosynthesis-antenna proteins.

Genes and proteins involved in capsanthin biosynthesis During fruit development, the content of capsanthin was found to be very low in the IMG and MG stages, gradually increasing in the Br and MR stages (Figure 7-A). As a type of carotenoid, 16


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capsanthin is mainly synthesized in fruit of red peppers64, and accumulates in the thylakoid membrane of mature fruit pulp plastid65. Although the transcript expression of many genes in the capsanthin biosynthetic pathway was significantly changed during fruit development, abundances of the corresponding proteins were very low or undetectable (Figure 7-C). Phytoene synthase (PSY) is a key enzyme in the carotenoid biosynthetic pathway66, and is up-regulated during fruit coloration21,67. In this study, transcripts of the PSY (Capana04g002519) gene began to increase significantly at the Br stage, while surprisingly the protein abundance of PSY showed no significant difference during fruit development. The underlying causes of PSY transcript and protein abundance difference during pepper fruit development need to be further studied. Nonetheless, this suggests that PSY might not be directly involved in the regulation of capsanthin content in pepper fruit.

Figure 7 β-carotene hydroxylase is a downstream gene in the capsanthin biosynthetic pathway, and has two paralogs, Crtz-1 and Crtz-2. Crtz-2 is highly expressed in the late stage of fruit ripening, and only Crtz-2 plays a role in fruit color formation68. During pepper fruit development, 17



accumulation of Crtz-2 protein lagged behind its transcription. The transcript expression of Crtz-2 was increased significantly from the MG stage and reached a very high level at the Br and MR stages, while its protein abundances at the IMG, MG and Br stages were all at a low level, and only were increased at the MR stage by 1.35- and 4.76-fold in SJ11-3 and 06g19-1-1-1, respectively. The capsanthin/capsorubin synthase (CCS) gene is present on the membrane of the pepper peel69. In the red pepper fruit, capsanthin replaces chlorophyll, while in yellow fruit CCS is not expressed or has been mutated70. Ha et al.71 found that silencing of the CCS gene leads to reduced capsanthin biosynthesis, causing ripe fruit to turn yellow. The expression of CCS1 protein (Capana06g000615) was not only increased by 1786- and 1602-fold in the Br stage compared with the MG stage, but also continuously increased by 2.02- and 1.63-fold in the MR stage compared with the Br stage in SJ11-3 and 06g19-1-1-1, respectively. The abundant accumulation of both CCS1 transcripts and proteins at the Br and MR stages was consistent with pepper fruit color changes. At the same time, CCS1 protein levels were 0.94- and 0.68-fold higher at Br and MR stages, respectively, in 06g19-1-1-1 than in SJ11-3, also consistent with the higher capsanthin contents in 06g19-1-1-1 at these two stages. This indicates that CCS1 may play a critical role in the regulation of capsanthin synthesis. In previous studies, there has been less focus on the upstream geranylgeranyl diphosphate synthase (GGPS) proteins in the synthetic pathway. In sweet orange, up-regulation of GGPS protein increased substrate content for lycopene accumulation34. We found that when some key downstream proteins in the pathway were expressed in high levels, GGPS1 (Capana04g000412) was also expressed in a high level (increased by 3.48- and 31.48-fold at the Br stage compared with the MG stage in SJ11-3 and 06g19-1-1-1, respectively), indicating that the content of capsanthin was also regulated by the upstream protein GGPS1. In addition, we found that same as the CCS1 protein, the expression of the GGPS1 protein in the two pepper varieties at the Br and MR stages also varied significantly. The level of GGPS1 protein was 2.11- and 1.11-fold higher at Br and MR stages, respectively, in 06g19-1-1-1 than in SJ11-3, further suggesting that it may be another important factor contributing to the difference in capsanthin contents between 18


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the two pepper varieties.

Genes and proteins involved in tocopherol biosynthesis Tocopherol is the main component of vitamin E and is synthesized in chloroplasts of higher plants72. We measured tocopherol contents during the pepper fruit development in the two varieties and found that there was a certain amount of tocopherol at the IMG stage and the contents were increased throughout the developmental process (Figure 7-B). Homogentisate phytyltransferase (HPT) is a membrane-bound protein present in chloroplasts and is a rate-limiting enzyme in the tocopherol biosynthetic pathway. Overexpression of the HPT gene can effectively increase the total amount of tocopherol73. By contrast, silencing HPT leads to a loss of tocopherol74. In our study, the expression HPT (Capana01g004250) was very low at both transcript and protein levels throughout the pepper fruit development (Figure 7-C). In addition, transcript

and

protein

levels

of

hydroxyphenylypruvate

dioxygenase

(HPPD)

and

2-methy-6-phytylbenzoquinine methytransferase (MPBQMT), two other genes/proteins related to tocopherol synthesis did not change significantly and their protein abundances were also maintained at extremely low levels (Figure 7-C). These results suggest that HPT, HPPD and MPBQMT might not be involved in the regulation of tocopherol accumulation in pepper fruits. The transcript levels of γ-tocopherol methyl-transferase (γ-TMT; Capana01g001103) was significantly up-regulated from MG to Br stages, and its protein levels were basically consistent with its transcript levels. In SJ11-3 and 06g19-1-1-1, the expression of γ-TMT protein was increased significantly by 2.43- and 1.48-fold, respectively, from MG to Br stage. γ-TMT can promote the conversion and synthesis of different tocopherol components, but has little effect on the total tocopherol content75,76. Kanwischer et al.77 found that the total amount of tocopherol in transgenic Arabidopsis leaves overexpressing the VTE1 gene, which encodes a tocopherol cyclase (TC), was nearly 7-fold higher than that of the control. In this study, expression of the TC (Capana08g000837) protein lagged behind the change in its transcription; the expression of the TC gene was significantly up-regulated from the Br to MR stage, and its expression at the Br 19



stage was 1.33-fold higher in 06g19-1-1-1 than in SJ11-3. The changes in expression of the TC protein at different stages of the two pepper varieties were basically consistent with the changes in tocopherol contents. At the same time, tocopherol biosynthesis is limited to some extent by upstream synthetic pathways78. GGDP is not only a precursor product of the carotenoid biosynthetic pathway, but also a common metabolite of the tocopherol biosynthetic pathway. The significant increase in GGPS1 protein abundance during color change and red pepper ripening stage resulted in a large increase in the GGDP substrate. But the increase in tocopherol content during the color change and red pepper ripening stages was mild, indicating that its effect on tocopherol synthesis was minor79,80. Production of PDP by GGDP is mediated by the catalytic action of geranylgeranyl diphosphate reductase (GGR), and PDP is the necessary precursor for the synthesis of tocopherol. In this study, the changes in transcript and protein levels of GGR (Capana03g000791) were opposite to several downstream genes/proteins in the pathway. For example, the protein was significantly down-regulated by 1.1-fold in SJ11-3 from IMG to MG and by 1.97-fold in 06g19-1-1-1 from Br to MR (Figure 7-C). High abundance of GGR protein in early stages of pepper fruit development could promote the production of PDP by GGDP, which led to the production of tocopherol in IMG and MG stages. A large increase in the abundance of GGPS1 protein in the middle and late stages of fruit development could promote the large-scale synthesis of GGDP. By contrast, decrease in the abundance of GGR could result in a decrease in the content of PDP, which further led to the slowed down of tocopherol synthesis at later stages. Therefore, the biosynthesis of tocopherol in the development of pepper fruit may be mainly co-regulated by upstream GGR and downstream TC proteins. GGPS1 and GGR jointly regulate the distribution of GGDP substrate, and a small amount of GGDP substrate in early stages of development is mainly used for biosynthesis of tocopherol, and a large increase in GGDP substrate in the later stage is mainly used for biosynthesis of capsanthin.

Genes and proteins involved in ascorbate biosynthesis 20


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The ascorbate content gradually increased during pepper fruit development with the greatest increase seen from MG to Br (Figure 8-A). However, the transcript and protein levels of related genes in the L-galactose pathway were decreased or maintained at low levels during the development of pepper fruit (Figure 8-B). In brief, most of the genes had higher transcript levels at early stages of fruit development, showing basically similar levels at IMG and MG stages and decreased gradually in the later stages, especially at the Br stage. The changes of protein levels were less significant than transcript levels, but their changes mainly occurred before the Br stage, indicating that the synthesis of ascorbate in pepper fruit was mainly completed before the color-changing stage, and reached dynamic balances between accumulation and metabolism in later stages81, which is similar to ascorbate synthesis in peach and tomato fruits82,83.

Figure 8 Numerous

studies

have

shown

that

glucose-6-phosphate

isomerase

(PGI),

mannose-6-phosphate isomerase (PMI), phosphomannomutase (PMM), L-galactono-1,4-lactone 21



dehydrogenase (GLDH) and L-galactose dehydrogenase (GalDH) are not the rate-limiting enzymes for ascorbate synthesis84. The main factors affecting the synthesis of ascorbate may be the transcription of GDP-L-galactose phosphorylase (GGP) (or the synergistic effect of GDP-mannose-3,5-epimerase (GME) and GGP), and the feedback of GGP85. GGP is the key rate limiting enzyme of L-galactose pathway in Arabidopsis and tobacco86,87. Its 5’ untranslated region contains a highly conserved upstream open reading frame (uORF). At high ascorbate concentration, the translation of uORF inhibits GGP translation, while at low concentration, translation of GGP inhibits translation of uORF88. In this study, GGP was highly transcribed in pepper fruit during the entire developmental process, but no GGP protein was identified. In addition, GME protein was significantly down-regulated (by 1.48 fold) from Br to MR in SJ11-3, and GDP-mannose pyrophosphorylase (GMP) was significantly down-regulated (by 5.8 fold) from MG to Br in 06g19-1-1-1. We speculate that the pepper GGP protein may regulate the ascorbate synthesis in very early stages of fruit development (before the IMG stage), and the ascorbate synthesis was then regulated by the GMP protein in later stages. Because GMP protein levels were significantly different between the two pepper varieties (4.31 fold lower at the Br stage and 2.8 fold lower at the MR stage in 06g19-1-1-1 than in SJ11-3), which was consistent with the ascorbate contents in Br and MR stages of the two pepper varieties.

Real time PCR and parallel reaction monitoring (PRM) validation qRT-PCR experiments were performed on 12 selected genes using gene-specific primers. Transcript abundances were calculated over the entire course of pepper fruit development. qRT-PCR analysis showed a similar trend of transcript abundance to that determined by RNA-Seq, confirming the reliability of our RNA-Seq data. Proteins of these 12 genes were also selected for quantitative verification of protein abundance by PRM. PRM verification enabled the detection of Capana10g002320 (LCYB) with the abundance increased significantly at Br and MR stage (Figure 9), which was consistent with the increase of the capsanthin content, but not detected by label-free quantitation in most of the developmental stages in the two pepper 22


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varieties. However, 11 of the 12 (91.7％) proteins showed similar trends of abundance between PRM and label-free quantitation. Thus, protein abundances derived from the label-free proteome data were highly reliable.

Figure 9 Conclusions We identified 23,349 transcripts and 5,455 protein groups at different fruit developmental stages in two varieties of pepper using RNA-Seq and label-free quantitation technologies. To our knowledge, this is the first study to apply the combined transcriptome and proteome analysis to the development of pepper fruits. In this study, integration of the transcriptome and proteome profiles uncovered a number of candidate transcripts/proteins that might be involved in capsanthin, tocopherol, and ascorbate biosynthesis and cell wall metabolism during fruit 23



development and ripening. In peppers, the synthesis of capsanthin is mainly regulated by key proteins especially GGPS1 and CCS1. The synthesis of tocopherol is mainly regulated by proteins GGPS1, GGR and TC. GGPS1 is involved not only in the regulation of capsanthin synthesis, but also in the regulation of tocopherol synthesis. In early stages of pepper fruit development, the catalytic products of GGPS1 are mainly used in the synthesis of tocopherol, while in later stages, they are mainly used in the synthesis of capsanthin. The synthetic pathway of ascorbate is mainly regulated by proteins GGP and GMP. This study provides extensive new information on the transcriptome, proteome and the correlation of transcriptome and proteome during pepper fruit development. Analysis of this resource has enabled us to examine mechanisms for transcript and protein diversification, which expands the knowledge of transcriptome and proteome complexity in non-climacteric species.

Conflict of interest The authors declare no conflicts of interest.

Acknowledgments This work was financially supported by the National Key Research and Development Program of China (2016YFD0101704).

24


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Supporting information Table S-1 List of correlated and differentially expressed correlated genes/proteins in different compared stages. Table S-2 Primers used for qRT-PCR in this study. Table S-3 Peptide sequences used for PRM in this study. Table S-4 Expression profiles of pepper genes during fruit development. Table S-5 Pepper peptide intensity data during fruit development. Table S-6 Pepper protein intensity data during fruit development. Table S-7 Differentially expressed transcripts of pepper fruit. Table S-8 Differentially expressed proteins of pepper fruit. Table S-9 List of proteins with change patterns at three key color-changing stages. Methods S-1 Transcriptome analyses. Methods S-2 Proteome analyses. Figure S-1 Peptide quality statistics. (A) Andromeda score distribution of peptide fragments. (B) Protein relative molecular mass distribution. (C) Peptide segment quantitative distribution. (D) Peptide sequence length distribution. (E) Peptide sequence coverage distribution. Figure S-2 Analysis of repeatability test of fruit transcriptome and proteome. A and B represent SJ11-3 and 06g19-1-1-1,respectively; 1, 2, 3, and 4 represent IMG, MG, Br and MR stage respectively; a, b and c represent replicates in a sample. Figure S-3 Correlation between transcript and protein abundance ratios between consecutive developmental stages of all quantified transcript-protein pairs (only those with fold change >2 and P-value ≤ 0.05 are shown). A, B and C represent MG/IMG, Br/MG and MR/Br of the SJ11-3 fruit, respectively. D, E and F represent MG/IMG, Br/MG and MR/Br of the 06g19-1-1-1 fruit, respectively.

25



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Page 32 of 45

Table 1 Correlation of all quantified transcripts and proteins in different comparisons. SJ11-3

06g19-1-1-1

MG/IMG

Br/MG

MR/Br

MG/IMG

Br/MG

MR/Br

Transcripts

17658

16010

16002

17411

16350

15883

Proteins

3407

2967

2792

3014

2659

2493

Correlated number

3335

2846

2663

2945

2582

2392

Up

711

3546

573

717

1946

563

Down

1616

4614

1209

779

3306

1161

Up

77

148

136

89

264

130

Down

104

260

132

104

370

178

Up

19

110

30

22

117

11

Down

21

162

40

17

127

45

DE_Transcript

DE_Protein

DE_Correlated

Number of transcripts, proteins, and correlated transcripts and proteins in consecutive developmental stages (MG/IMG, Br/MG and MR/Br) and numbers of differentially expressed transcripts and proteins are given (fold change >2 and P-value ≤ 0.05). DE, differentially expressed. IMG, immature green; MG, mature green; Br, breaker; MR, mature red

32


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Figure legends Figure 1. Fruits of the two pepper (Capsicum annuum L.) varieties at different stages of development used in this study. IMG, immature green (20 DAA); MG, mature green (30 DAA); Br, breaker (40 DAA); MR, mature red (50 DAA). A, SJ11-3. B, 06g19-1-1-1.

Figure 2. Venn diagrams showing transcriptome and proteome coverage in different sampling stages. Number of transcripts and proteins identified and quantified in each sample in at least two of three replicates is shown. A, transcripts; B, proteins. IMG, immature green; MG, mature green; Br, breaker; MR, mature red.

Figure 3. Principal component analysis (PCA) of transcript and protein profiles.

Figure 4. Top 20 GO categories assigned to the differentially expressed correlated proteins. The correlated proteins were categorized based on gene ontology annotation and the proportion of each category is displayed in the categories of biological process (BP), molecular function (MF), and cellular component (CC).

Figure 5. Heat map of 45 fruit enlargement and ripening related transcripts and proteins during pepper fruit development. Pearson correlation coefficients (cc) between transcripts and proteins in SJ11-3 and 06g19-1-1-1 are shown, with significant positive correlations marked with *, and high positive (r >0.7) and negative correlations marked in red and blue, respectively.

Figure 6. Abundance pattern of correlated proteins at three key color-changing stages. A, Abundance pattern of correlated proteins classified into nine patterns during fruit maturation (MG-Br-MR). These nine patterns are stable, continuous increase, early increase, increase-decrease, late increase, continuous decrease, early decrease, decrease-increase and late decrease. B, Patterns of protein abundance summarized according to the top 30 KEGG pathways. 33



Note that the numbers (left and right) and percentage (bottom) of proteins in the color-coded MapMan functional classes are shown. PPER, Protein processing in endoplasmic reticulum; PS, photosynthesis; Va. Le & iso, Valine, leucine and isoleucine; Gly. Ser & thr, Glycine, serine and threonine; Ami & nucle sugar, Amino sugar and nucleotide sugar; CFPO, Carbon fixation in photosynthetic organisms; Ala. asp & glu, Alanine, aspartate and glutamate; meta, metabolism; bio, biosynthesis.

Figure 7. Comparative analysis of carotenoid and tocopherol metabolic genes and proteins from the IMG stage to the MR stage. A, Capsanthin contents in different developmental stages of pepper fruit. B, Tocopherol contents in different developmental stages of pepper fruits. C, Metabolic pathway of capsanthin and tocopherol. Transcript and protein levels are represented by fruit of SJ11-3 (SJ; red arrowheads) and 06g19-1-1-1 (06g; blue arrowheads), and variation in the levels is indicated by the different shades of the arrowheads and arrows. De., Decrease; In., increase. Three experimental replicates were used to indicate up-regulation (up arrow) or down-regulation (down arrow). The horizontal lines represent no significant changes. The left side of the black vertical line represents the transcriptional analysis, and the right side of the black vertical line represents the protein analysis. a, b and c represents MG/IMG, Br/MG and MR/Br, respectively. GGPS, geranylgeranyl diphosphate synthase; PSY, phytoene synthase; PDS, phytoene desaturase; ZDS, ζ-carotene desaturase; CrtISO, carotene isomerase; LCYB, lycopene beta-cyclase; LCYE, lycopene ε-cyclase; Crtz-2, beta-carotene 3-hydroxylase; ZEP, zeaxanthin epoxidase; VDE, violaxanthin deepoxidase; CCS, capsanthin/capsorubin synthase; LUT1, carotene epsilon-monooxygenase. GGR, geranylgeranyl diphosphate reductase; HPPD, hydroxyphenylypruvate dioxygenase; HPT, homogentisate phytyltransferase; MPBQMT, 2methy- 6- phytylbenzoquinine methytransferase; TC, tocopherol cyclase; γ-TMT, γ-tocopherol methyl-transferase.

Figure 8. Comparative analysis of ascorbate metabolic genes and proteins from the IMG stage to 34


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the MR stage. A, Ascorbate contents in different developmental stages of pepper fruit. B, Metabolic pathway of ascorbate. Transcript and protein levels are represented by fruit of SJ11-3 (SJ; red arrowheads) and 06g19-1-1-1 (06g; blue arrowheads), and variation in the levels is indicated by the different shades of the arrowheads and arrows. De., Decrease; In., increase. Three experimental replicates were used to indicate up-regulation (up arrow) or down-regulation (down arrow). The horizontal lines represent no significant changes. The left side of the black vertical line represents the transcriptional analysis, and the right side of the black vertical line represents the protein analysis. a, b and c represents MG/IMG, Br/MG and MR/Br, respectively. HK, hexokinase; PGI, Glucose-6-phosphate isomerase; PMI, mannose-6-phosphate isomerase; PMM,

phosphomannomutase;

GDP-mannose-3,5-epimerase;

GMP, GGP,

L-galactose-1-phosphate

phosphatase;

L-galactono-1,4-lactone

dehydrogenase;

GDP-mannose

pyrophosphorylase;

GME,

phosphorylase;

GPP,

dehydrogenase;

GLDH,

GDP-L-galactose GalDH, APX,

L-galactose ascorbate

peroxidase.

MDHAR,

monodehydroascorbate reductase; MIOX, myo-inositol oxygenase.

Figure 9. Validation and expression analysis of selected genes and proteins using qRT-PCR and PRM. The ordinate (left) represents the relative expression of genes; The ordinate (right) represents the expression of proteins. Capana03g000054, PDS; Capana04g002519, PSY; Capana10g002320,

LCYB;

Capana04g000412,

GGPS1;

Capana03g000791,

GGR;

Capana03g002170, Capana08g000837, Capana08g001246,

Crtz-2;

Capana06g000615,

CCS1;

TC;

Capana01g001103,

TMT;

GME;

Capana08g001512,

XTH1;

Capana09g002242, XTH32. IMG, immature green; MG, mature green; Br, breaker; MR, mature red.

35



For TOC only

36


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Figure 1. Fruits of the two pepper (Capsicum annuum L.) varieties at different stages of development used in this study. IMG, immature green (20 DAA); MG, mature green (30 DAA); Br, breaker (40 DAA); MR, mature red (50 DAA). A, SJ11-3. B, 06g19-1-1-1.



Figure 2. Venn diagrams showing transcriptome and proteome coverage in different sampling stages. Number of transcripts and proteins identified and quantified in each sample in at least two of three replicates is shown. A, transcripts; B, proteins. IMG, immature green; MG, mature green; Br, breaker; MR, mature red. 137x105mm (300 x 300 DPI)


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Figure 3. Principal component analysis (PCA) of transcript and protein profiles. 229x97mm (300 x 300 DPI)



Figure 4. Top 20 GO categories assigned to the differentially expressed correlated proteins. The correlated proteins were categorized based on gene ontology annotation and the proportion of each category is displayed in the categories of biological process (BP), molecular function (MF), and cellular component (CC). 176x115mm (300 x 300 DPI)


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Figure 5. Heat map of 45 fruit enlargement and ripening related transcripts and proteins during pepper fruit development. Pearson correlation coefficients (cc) between transcripts and proteins in SJ11-3 and 06g19-11-1 are shown, with significant positive correlations marked with *, and high positive (r >0.7) and negative correlations marked in red and blue, respectively. 114x68mm (300 x 300 DPI)



Figure 6. Abundance pattern of correlated proteins at three key color-changing stages. A, Abundance pattern of correlated proteins classified into nine patterns during fruit maturation (MG-Br-MR). These nine patterns are stable, continuous increase, early increase, increase-decrease, late increase, continuous decrease, early decrease, decrease-increase and late decrease. B, Patterns of protein abundance summarized according to the top 30 KEGG pathways. Note that the numbers (left and right) and percentage (bottom) of proteins in the color-coded MapMan functional classes are shown. PPER, Protein processing in endoplasmic reticulum; PS, photosynthesis; Va. Le & iso, Valine, leucine and isoleucine; Gly. Ser & thr, Glycine, serine and threonine; Ami & nucle sugar, Amino sugar and nucleotide sugar; CFPO, Carbon fixation in photosynthetic organisms; Ala. asp & glu, Alanine, aspartate and glutamate; meta, metabolism; bio, biosynthesis. 207x126mm (300 x 300 DPI)


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Figure 7. Comparative analysis of carotenoid and tocopherol metabolic genes and proteins from the IMG stage to the MR stage. A, Capsanthin contents in different developmental stages of pepper fruit. B, Tocopherol contents in different developmental stages of pepper fruits. C, Metabolic pathway of capsanthin and tocopherol. Transcript and protein levels are represented by fruit of SJ11-3 (SJ; red arrowheads) and 06g19-1-1-1 (06g; blue arrowheads), and variation in the levels is indicated by the different shades of the arrowheads and arrows. De., Decrease; In., increase. Three experimental replicates were used to indicate up-regulation (up arrow) or down-regulation (down arrow). The horizontal lines represent no significant changes. The left side of the black vertical line represents the transcriptional analysis, and the right side of the black vertical line represents the protein analysis. a, b and c represents MG/IMG, Br/MG and MR/Br, respectively. GGPS, geranylgeranyl diphosphate synthase; PSY, phytoene synthase; PDS, phytoene desaturase; ZDS, ζ-carotene desaturase; CrtISO, carotene isomerase; LCYB, lycopene beta-cyclase; LCYE, lycopene ε-cyclase; Crtz-2, beta-carotene 3-hydroxylase; ZEP, zeaxanthin epoxidase; VDE, violaxanthin deepoxidase; CCS, capsanthin/capsorubin synthase; LUT1, carotene epsilon-monooxygenase. GGR, geranylgeranyl diphosphate reductase; HPPD, hydroxyphenylypruvate dioxygenase; HPT, homogentisate phytyltransferase; MPBQMT, 2- methy- 6- phytylbenzoquinine methytransferase; TC, tocopherol cyclase; γTMT, γ-tocopherol methyl-transferase. 161x114mm (300 x 300 DPI)



Figure 8. Comparative analysis of ascorbate metabolic genes and proteins from the IMG stage to the MR stage. A, Ascorbate contents in different developmental stages of pepper fruit. B, Metabolic pathway of ascorbate. Transcript and protein levels are represented by fruit of SJ11-3 (SJ; red arrowheads) and 06g191-1-1 (06g; blue arrowheads), and variation in the levels is indicated by the different shades of the arrowheads and arrows. De., Decrease; In., increase. Three experimental replicates were used to indicate up-regulation (up arrow) or down-regulation (down arrow). The horizontal lines represent no significant changes. The left side of the black vertical line represents the transcriptional analysis, and the right side of the black vertical line represents the protein analysis. a, b and c represents MG/IMG, Br/MG and MR/Br, respectively. HK, hexokinase; PGI, Glucose-6-phosphate isomerase; PMI, mannose-6-phosphate isomerase; PMM, phosphomannomutase; GMP, GDP-mannose pyrophosphorylase; GME, GDP-mannose-3,5-epimerase; GGP, GDP-L-galactose phosphorylase; GPP, L-galactose-1-phosphate phosphatase; GalDH, L-galactose dehydrogenase; GLDH, L-galactono-1,4-lactone dehydrogenase; APX, ascorbate peroxidase. MDHAR, monodehydroascorbate reductase; MIOX, myo-inositol oxygenase. 101x106mm (300 x 300 DPI)


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Figure 9. Validation and expression analysis of selected genes and proteins using qRT-PCR and PRM. The ordinate (left) represents the relative expression of genes; The ordinate (right) represents the relative expression of proteins. Capana03g000054, PDS; Capana04g002519, PSY; Capana10g002320, LCYB; Capana03g002170, Crtz-2; Capana06g000615, CCS1; Capana04g000412, GGPS1; Capana08g000837, TC; Capana01g001103, TMT; Capana03g000791, GGR; Capana08g001246, GME; Capana08g001512, XTH1; Capana09g002242, XTH32. IMG, immature green; MG, mature green; Br, breaker; MR, mature red. 91x73mm (300 x 300 DPI)


Integrative transcriptome and proteome analysis identifies major

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