Comparative proteomic analysis reveals that chlorophyll metabolism

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Comparative proteomic analysis reveals that chlorophyll metabolism contributes to leaf color changes in wucai (Brassica campestris L.) responding to cold acclimation Shilei Xie, Libing Nie, Yushan Zheng, Jie Wang, Mengru Zhao, Shidong Zhu, Jinfeng Hou, Guohu Chen, Chenggang Wang, and Lingyun Yuan J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.9b00016 • Publication Date (Web): 30 Apr 2019 Downloaded from http://pubs.acs.org on April 30, 2019

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Journal of Proteome Research

Comparative

proteomic

analysis

reveals

that

chlorophyll metabolism contributes to leaf color changes in wucai (Brassica campestris L.) responding to cold acclimation Shilei Xie 1,2, Libing Nie1,2, Yushan Zheng1,2, Jie Wang1,2, Mengru Zhao1,2, Shidong Zhu1,2,3, Jinfeng Hou1,2,3, Guohu Chen1,2, Chenggang Wang1,2,3,*, Lingyun Yuan1,2,3,*

1College

of Horticulture, Vegetable Genetics and Breeding Laboratory, Anhui Agricultural

University, 130 West Changjiang Road, 230036 Hefei, Anhui, China; 2Provincial

Engineering Laboratory for Horticultural Crop Breeding of Anhui, 130 West of

Changjiang Road, 230036 Hefei, Anhui, China; 3Wanjiang

Vegetable Industrial Technology Institute, Maanshan, Anhui, 238200, China

1

ACS Paragon Plus Environment

Journal of Proteome Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract Chlorophyll is a vital photosynthetic pigment that plays a key role in plant development, participating in light energy capture and energy conversion. In this study, a novel wucai (Brassica campestris L.) germplasm with green outer leaves and yellow inner leaves at the adult stage (W7-2) was used to examine chlorophyll metabolism response to cold acclimation. A green leaf wucai genotype without leaf color changes named W7-1 was selected as the control to evaluate the chlorophyll metabolism changes of W7-2. Compared to W7-1, the contents of chlorophyll a (Chl a) and chlorophyll b (Chl b) in W7-2 were significantly reduced at five developmental stages (13, 21, 29, 37 and 45 days after planting (DAP)). An iTRAQ-based quantitative proteomic analysis was carried out at 21 and 29 DAP according to the leaf color changes in both of genotypes. 1409 proteins were identified, while 218 of them displayed differential accumulations between W7-2 and W7-1 during the two developmental stages. The differentially expressed proteins (DEPs) mainly assigned to chlorophyll biosynthesis, photosynthesis, carbohydrate metabolism, ribosome metabolism and posttranslational modification. Among these DEPs, NADPH-protochlorophyllide oxidoreductase (PORB) and Mg-protoporphyrin IX chelatase 1 (CHLI1) were the key enzymes participating in chlorophyll (Chl) biosynthesis, which was down-regulated at 21 DAP and up-regulated at 29 DAP in W72 compared with W7-1, respectively The expression analysis of genes of three subunits of Mgchelatase (CHLI1, CHLD and CHLH), Genomes Uncoupled 4 (GUN4) and Thioredoxin (TRX3) associated with chlorophyll metabolism also displayed significant down-regulation in W7-2. In particular, PORB showed significant up-regulation in W7-2, significantly affecting chlorophyll biosynthesis. Additionally, differences in chlorophyll metabolism between W7-2 and W7-1 were in terms of altered photosynthesis, carbohydrate and energy metabolism. We found that the transcription levels of most photosynthesis proteins showed significantly lower levels, and the genes expression level, associated with carbohydrate and energy metabolism, were lower in W7-2 than in W7-1. Therefore, the present study results help understand the physiological and molecular mechanisms underlying leaf coloring responding to cold acclimation. Keywords: Wucai; Leaf color; iTRAQ-based quantitative proteomics; Chlorophyll biosynthesis; Mg-protoporphyrin IX chelatase 1; NADPH-protochlorophyllide oxidoreductase

2


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Abbreviations Chl

Chlorophyll

Glu

Glutamic acid

ALA

5-aminolevulinic acid

PBG

Porphobilinogen

Proto IX

Protoporphyrin IX

Mg-proto IX

Mg-protoporphyrin IX

Pchlide

Protochlorophyllide

GluTR

Glutamyl-tRNA reductase

PORB

NADPH-protochlorophyllide oxidoreductase

CHLI1

Mg-protoporphyrin IX chelatase 1

GUN4

Genomes Uncoupled 4

TRX3

Thioredoxin

DEPs

Differentially expressed proteins

iTRAQ

Isobaric Tags for Relative and Absolute Quantitation

PGK1

Phosphoglycerate kinase

MDH2

Malate dehydrogenase

FBA1

Fructose-bisphosphate aldolase

rbcL

Ribulose bisphosphate carboxylase

UDP1

UTP-glucose-1-phosphate uridylyl transferase

CAB

Chlorophyll a/b binding protein

3



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Introduction Chl molecules are universal in photosynthetic organisms. In an antenna system, Chl harvests light energy and drives electron transfer in the reaction center.1 The regular chlorophyll biosynthesis and degradation are important for all photosynthetic microorganisms and photoautotrophic plants.24 In Arabidopsis thaliana, 18 enzymes, encoded by 29 genes, could catalyze chlorophyll biosynthesis starting from glutamate.5,6 Photosynthetic efficiency and carbon fixation are closely related to Chl content and the morphology and structure of chloroplasts.7 There are a number of Chl-deficient mutants that have been identified in Arabidopsis8, Brassica napus9, barley10 and wheat11. Among the many mutant types, there is a special type of leaf color variation “low temperature sensitive leaf color mutant”. This type of mutant exhibits normal or near-normal leaf color at normal temperatures, but exhibits a mutational leaf color at lower temperatures.12 Several temperature-sensitive leaf color mutant genes were found in rice. v1, v2, v3 and tcd5 are identified as low temperature albino mutant that have albino leaf under low temperature (20℃) and normal green leaves under the higher temperature (32℃).13 The wandering jew (Tradescantia fluminensis) also has a thermalsensitive leaf-color mutant (mt). Its young leaf color of the mutant is pink at low temperature (6-20℃) and become green at normal temperature (20-35℃). The lower surface leaf of the mutant still remains purple color. However, the wild-type young leaf phenotype remains green under low temperature conditions.14 The leaf color phenotype of the wheat mutant, designated as ‘stage albinism line of winter wheat’ FA85, performs gradual albinism of all aboveground parts at low temperature. As the temperature increased, the aboveground part of FA85 turns to green again.15 The proteomic approach has been used to identify Chl biosynthesis-related proteins and to study the molecular mechanism of the celery under temperature stress. Identification of Chl biosynthesis-related

proteins,

including

Mg-chelatase

and

glutamate-1-semialdehyde

aminotransferase, were found to be up-regulated in celery under cold stress. At the same time, the content of Chl in celery leaf was decreased under high temperature and low temperature.16 In the present study with a wucai yellow inner leaf phenotype, the inner leaves of this phenotype are green in seedlings at the early autumn temperatures (2433°C); as the plant grows, the inner leaves turn to yellow in the adult stage under low-temperature (818°C). Although the inner 4


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leaves turn yellow, this phenotype can still maintain normal growth in winter, and this shows that leaf color changes do not affect the entire growth cycle. According to the study cited above, we believe that the yellow inner leaf phenotype may be an adaptation to cold acclimation. Wucai (Brassica campestris L. ssp. chinensis var. rosularis Tsen et Lee) is a subspecies of Chinese cabbage (Brassica pekinensis) and an important vegetable crop. The variety originated in China and is distributed mainly in the Yangtze-Huaihe River basin.17,18 After cold temperatures, its quality will be improved. Due to the various phenotypes and high nutritional value, wucai are valuable experimental material. In our previous study, a new germplasm wucai genotype named W7-2 was found having yellow inner leaves. The leaves are green at the seedling stage, whereas inner yellow leaves appear at the later developmental stages. To date, there has been very little research on leaf color conversion of wucai, and the conversion mechanism is still unknown. Proteomics is an efficient method to be used to study complicated protein populations in many species. Isobaric tags for relative and absolute quantitation (iTRAQ) is one of the most reliable techniques for quantifying proteins based on peptide labeling; This method can identify and accurately quantify proteins in multiple samples within the dynamic range of protein abundance.1922 To investigate the possible mechanism of color conversion in W7-2, iTRAQbased proteomics analysis was used to reveal differentially expressed proteins between W7-2 and W7-1 at two key developmental stages with leaf color changes. The Chl biosynthesis pathway, including several intermediate metabolites, was also compared between the two wucai genotypes. The transcription levels of genes related to Chl biosynthesis and photosynthesis were analyzed to elucidate the molecular mechanism of leaf color change. Materials and methods Plant materials Two wucai (Brassica campestris L.) cultivars (Fig. 1), here referred to as W7-2 and W7-1, were selected for the present study. W7-2, a new germplasm line, has inner yellow leaves at the adult stage. W7-1, selected as the control genotype, has green leaves during the entire growth and development period. Both cultivars have been bred through multiple generations of systems, with good quality and high yields under suitable conditions. Both cultivars were widely planted in the Yangtze-Huai River Basin in China. 5



Growth conditions The experiment was carried out in the breeding basement of Anhui Agriculture University Seedlings of the two genotypes were planted in a greenhouse. The greenhouse conditions were under conditions of 26 ± 2℃ (day) and 20 ± 1.5℃ (night) with relative humidity 7080%. The seedlings with 4~5 leaves were transplanted in stroma, then moved into an artificial climate chamber. The parameters were in accordance with the normal temperature (local weather) (Table S1) with a 10h/14h (day/night) cycle and light intensity at 300 μmol∙m−2∙s−1. According to days after planting (DAP), the third fully expanded young leaves from the center were sampled every four days after planting. The samples were immediately frozen in liquid nitrogen and kept at -80°C for physiological and biochemical measurements. Measurement of color values Leaf color values were measured using a Chroma meter (CR-400-C, Konica Minolta Sensing Americas, Inc., USA) on the upper surface of the leaf every four days after planting. Ten plants were measured, and the measurements were repeated three times in the same leaf position for the two phenotypes. The color data was analyzed by CIELAB color coordinate system. There are three main leaf color values: Value L*, Value a* and Value b*. Hue angle (H*): these were purple, red, orange, yellow, yellow green and blue-green on a scale from 0 to 180, where H*= 0, purple; H*= 90, yellow and H*= 180, blue-green. In addition, Chroma (C*) refers to the purity of the color; the higher the purity, the sharper the performance; the lower the purity, the less bleak the performance.23,24 Measurement of pigment content Total Chl were measured by method of Arnon25 with slight modification. 0.2 g fresh leaf was placed in ethanol: acetone: water at certain volume ratio for 24 h at approximately 4°C for chlorophyll extraction under dark conditions. The absorbance was determined with a UV-Vis spectrophotometer (TU1950, PERSEE, China) at wavelengths at 665 nm and 649 nm. The concentrations were calculated as (μg·mL-1): CChl a= 13.95×A665 - 6.88 ×A649; CChl b = 24.96 × A649 - 7.32 × A665. Protein preparation and iTRAQ labeling According to the comprehensive analysis results of color values and Chl content, the color values changed most significantly and Chl content decreased significantly from 21 DAP to 29 6


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DAP. So, the 21 DAP was considered as the green inner leaf stage and 29 DAP as the yellow inner leaf stage. Leaves of the two wucai genotypes at 21 DAP and 29 DAP were chosen for protein extraction by method of Isaacson26 and Yang27 with some modification and three biological replicates were taken. Protein digestion was carried out by the FASP procedure of Wisniewski28, and the method uses 8-plex iTRAQ reagents to label the resulting peptide mixture. One unit of iTRAQ reagent was dissolved in 24 μL isopropanol. The peptides labeled with the isobaric tags were incubated for 2 h at room temperature. The reaction was terminated by adding 200 μL water. Two phenotypes were labeled with the isobaric tags 113 (W7-1), 114 (W7-1), 115 (W7-2), and 116 (W7-2) at 21 DAP and 29 DAP, respectively. The samples were labeled as 113-1, 113-2, 113-3; 114-1, 114-2, 114-3; 115-1, 115-2, 115-3, 116-1, 116-2 and 116-3. All samples were multiplexed and vacuum dried. Peptide fractionation and quantitative proteomic analysis by LC-MS/MS To prepare for strong cationic exchange chromatography using an Agilent 1100 HPLC Purifier system (Agilent Technologies Inc., California, USA), the iTRAQ-labeled peptide mixtures were reconstituted and acidified in 4 mL of buffer A [25 mM KH2PO4 in 25% acetonitrile (ACN), pH 2.7] and loaded onto a 2.1 × 150 mm Agilent Zorbax Extend-C18 column. The peptide was eluted at a flow rate of 300 μL min-1 with a linear gradient of 5% buffer B (25 mM KH2PO4 and 1 M KCl in 25% ACN, pH 2.7) for 7 min, 560% buffer B for 20 min and 60100% buffer B for 2 min, and then maintained in 100% buffer B for 1 min. Elution was monitored by absorbance at 214 nm, and the fractions were collected every minute. The collected fractions were desalted using a C-18 column and were vacuum dried. Each fraction was resuspended were carried out at a flow rate 3 μL min-1 for 10 min with buffer C [acetonitrile (ACN)/formic acid (FA)/water = 2/0.1/98 (v/v/v)]. A total of 5 μL of supernatant was loaded onto the C18-reversed phase analytical column (75 μm × 15 cm, 3 μm, 120 Å, ChromXP Eksigent). The samples were loaded at 300 nL/min with a linear gradient of 5% buffer D (95% ACN, 0.1% FA) for 5 min, 335% buffer D for 35 min, 3560% buffer D for 35 min, 6080% buffer D for 2 min, 5% buffer D for 1 min, and finally maintained in 5% buffer D for 10 min. LC-MS/MS analysis was performed with a Q-Exactive mass spectrometer (Thermo Fisher Scientific) that was coupled to Easy-nLC (Thermo Fisher Scientific) according to the method 7



of Zhang29. Full-scans MS (300 - 1600 m/z) were performed in a mass resolution of 70000, and the AGC target value was set at 1e6. The ten most intense peaks in MS were fragmented with higher-energy collisional dissociation (HCD) with NCE of 30 in data-dependent positive ion mode. MS/MS spectra were obtained with a resolution of 17500 with an AGC target of 2e5 and a max injection time of 50 ms. Dynamic exclusion was set for 15 s. Protein identification and quantification MS/MS spectra were searched using Proteome DiscovererTM 2.2 (Thermo Scientific) run against the UniProt database30 (January 28, 2018, 172630 sequences), The search results were filtered with these options: significance threshold P < 0.05 (with 95% confidence) and ion score or expected cut-off < 0.05 (with 95% confidence). The main parameters are as follows: MS tolerance: 20 ppm MS/MS tolerance: 0.5 Da Enzyme: Trypsin Database: Brassica. fasta. Fixed modification: Carbamidomethyl (C), iTRAQ-8-plex (N-term), iTRAQ-8-plex (K). Variable modification: Oxidation (M). Acetyl (Protein N-term) Decoy database pattern: reverse Peptide FDR:0.01 Protein FDR:0.01 For protein quantification, the protein ratios were calculated as the median of only the unique peptides of the protein. All peptide ratios were normalized by the median protein ratio. Differentially expressed proteins (DEPs) were analyzed for significant up regulation or down regulation. One-sample t-tests were used to identify significant (P 1.2 or < 0.83 were considered to be differentially expressed as the average ratios of 115/113 and 116/114. Bioinformatic analysis of proteins Bioinformatic analysis of proteins was performed according to Yang27. The identified proteins were annotated by searching against the UniProt database. DEPs were grouped in line with biological functions using gene ontology (GO) terms (http://www.geneontology.org/) and 8


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Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.jp/kegg/) to determine the active biological pathways. Analysis of water-soluble carbohydrate and starch contents Freeze-dried leaf blades were used to determinate water-soluble carbohydrate and starch content, which were measured using a modified phenol-sulphuric acid method.31 Glucose content was measured using a Solarbio reagent kit (Cat #BC2500, Beijing Solarbio Science & Technology Co., Ltd, China). Measurement of chlorophyll metabolism Glutamic acid content Glutamic acid (Glu) content was measured using a Solarbio reagent kit (Cat #BC1580, Beijing Solarbio Science & Technology Co., Ltd, China). ALA content and PBG content The porphobilinogen (PBG) concentrations were determined according to the method of Bogorad32 and 5-aminolevulinic acid (ALA) concentrations was determined according to the method of Morton33 with slight modification. Proto IX, Mg-Proto IX and Pchlide contents The contents were measured by the method of Hodgins34 and Liu35 with some modifications. Fresh samples (0.3 g) were added to 5 ml of 80% alkaline acetone for grinding to a volume of 25 ml. The mixture was cultured in the dark until the tissue was bleached. Then, the mixture was centrifuged at 1500g for 10 min. The supernatant was extracted to determine the absorbance at 575 nm, 590 nm and 628 nm, respectively. Transcriptional expression analysis by quantitative RT-PCR To explore the expression patterns of related protein genes after the color conversion of W7-2 leaves, genes related to chlorophyll biosynthesis as well as photosynthesis proteins and carbohydrate metabolism-related genes were further analyzed by qRT-PCR. Total RNA samples were isolated from two different days after planting of wucai leaves using a total RNA kit (Takara Biomedical Technology Co., Beijing, China). The reverse transcription followed as the report of Cheng36. The BnaActin gene was used as the control gene. Gene-specific primers were designed using Primer Software Version 5.0 (Premier Biosoft International, CA, USA) (Table S2). The qRT-PCR amplification was performed as the method of Cao37. The relative 9



expression of gene was calculated using the 2-△△CT method.38 Statistical analysis Data were expressed as mean ± SD with at least three biological replicates. The difference was analyzed with SPSS 22.0 (SPSS Institute Inc., USA), and means were compared by Tukey’s test at significance level of 0.05. The related figures were drawn using GraphPad Prism software (GraphPad, USA). Results Analysis of leaf color parameter changes during the growth period The values of color parameters (color L*, color a* and color b*) varied with wucai leaves’ color changes during the growth and development periods (Table S3). Compared with W7-1, W7-2 had significantly higher color L* (lightness) and color b* (yellowness) values. In W7-2, color b* (yellowness) values and color L* (lightness) values also displayed remarkable increases with increasing number of days after planting, especially color b*(yellowness), which changed from 23.43 to 36.73 from 21 DAP to 29 DAP. There were no significant differences in color a*(greenness) values; this varied with days after planting in W7-2, while it was slightly increased in W7-1. During the growth period, little difference in the color L*(lightness) values was found in W7-1. There were no obvious differences in H* value with increasing days after planting in W7-1, whereas this was significantly increased in W7-2 (Fig. 2A). Chroma (C*) value changes were similar to Hue angle (H*) value changes in both genotypes (Fig. 2B). Phenotypic characterization of W7-2 and W7-1 When growing in an open field, W7-2 exhibited the normal green leaf phenotype from the seedling stage and displayed yellow inner leaves at the adult stage. To confirm the yellow inner leaves phenotype, the Chl levels under different days after planting were estimated at the growth stage. According to Chroma (C*) changes (Fig. 2A), five periods of 13, 21, 29, 37 and 45 DAP (Fig. 1C) were selected when the leaf color showed the largest changes. The Chl a and Chl b contents were observably higher in W7-1 than in W7-2 at all stages. This result was consistent with the previous color value (Fig. 3). However, Chl a and Chl b in W7-2 were significantly decreased in several stages, by 66.6% and 74.7%, respectively, at 45 DAP compared with 13 DAP, implying that the abnormal leaf color of W7-2 was attributed to decreased pigment content. In contrast, Chl a and Chl b were not significantly different or were slightly increased 10


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at the later stages in W7-1. Chl a and Chl b contents were increased by 53.4 % and 19.0 % in W7-1 at 45 DAP compared with 13 DAP, respectively. The Chl a:b ratio of W7-2 was higher than in W7-1 at later stages (Fig. 3D). Quantitative identification of leaf proteins using iTRAQ Total proteins in the leaves of the two wucai phenotypes were extracted from W7-2 and W7-1 at 21 and 29 DAP. A total of 1,014,954 peptides, 92,802 unique peptides and 5382 proteins (score sequence HT > 0 and unique peptides ≥ 1) were identified (Fig. 4). Proteins with FC > 1.2 (P < 0.05) were considered up-accumulated, whereas those with FC < 0.833 (P < 0.05) were considered as down-accumulated. Thus, 190 DEPs were detected in W7-2 and/or W7-1. Among the differentially expressed proteins identified in W7-2 compared to W7-1 at 21 and 29 DAP, 53 and 35 proteins were down-regulated at 21 DAP and 29 DAP, respectively. However, the levels of 77 and 53 proteins were up-regulated at 21 DAP and 29 DAP, respectively (Fig. 5A). Twenty differentially expressed proteins were shared at both 21 and 29 DAP (Fig. 5B). Furthermore, two proteins were up-accumulated at 21 DAP but were downaccumulated at 29 DAP. By contrast, one protein was down-accumulated at 21 DAP but was up-accumulated at 29 DAP, although this was an uncharacterized protein (Table S4). Bioinformatic analysis of differentially expressed protein species identified by iTRAQ To understand the function of the differentially expressed proteins, all quantified proteins were searched through the UniProt-GOA database, and GO annotation was carried out. Categories, cellular compartment, biological process and molecular function by GO annotation were assigned among all the DEPs (Fig. S1). A total of 218 differentially expressed proteins between W7-2 and W7-1 at 21 DAP and 29 DAP were classified into the 60 most significant functional groups, including molecular functions, cellular components and biological processes accounted for 20 GO terms, respectively. Based on the GO analysis, photosynthesis and generation of precursor metabolites and energy were the two major groups in terms of biological processes at 21 DAP. This indicates that photosynthesis is very easily affected in response to leaf color change. Protein domain specific binding and pigment binding were the two major molecular functional groups. Plastids and chloroplasts were the top two cellular compartments. However, in terms of biological processes, translation and peptide metabolic processes were the two major groups, while plastid 11



stroma and ribosomes were the two major molecular functional groups. The analysis of the heat map of the DEPs expression pattern in Fig. 6. To investigate the biological functions of these proteins, the analysis of KEGG pathway enrichment was also performed (Fig. 7). The most representative way is photosynthesis, followed by photosynthesisantenna proteins, metabolic pathways and carbon fixation in photosynthetic organisms at 21 DAP (Fig. 7A). At 29 DAP, KEGG terms including ribosome were highly enriched in DEPs (Fig. 7B). Based on our data, W7-2 inner leaf color formation had its own unique features. Before and after W7-2 leaf color changes, there were two modules of color metabolism pathways, the Chlorophyll a/b binding protein (CAB) metabolism pathway and the chlorophyll metabolism pathway. The Photosystem I complex and CAB were down-regulated at 21 DAP, and the CAB was also down-regulated at 29 DAP. PORB and CHLI1 proteins were related to the chlorophyll metabolism pathway. PORB was down-regulated at 21 DAP, while CHLI1 was up-regulated at 29 DAP. These are key proteins involved in chlorophyll metabolism. Chlorophyll synthesis pathway intermediate analysis In order to explore the time course of the change in color compounds conversion, we analyzed intermediate contents involved in the chlorophyll biosynthesis. Several important upstream and downstream intermediates contents in the chlorophyll biosynthesis were measured. The Glu, ALA, PBG, Proto IX, Mg-proto IX and Pchlide levels were estimated in both W7-1 and W7-2. As shown in Fig. 8, there was no significant difference in Glu content in W7-1 at different days after planting, while the level of Glu was significantly decreased in W7-2 from 13 to 29 DAP (Fig. 8A). W7-2 had significantly decreased levels of ALA, PBG, Proto IX, Mg-proto IX and Pchlide, by 72.5%,74.0%, 63.5%, 66.0% and 70.6%, respectively, at 45 DAP compared to 13 DAP. However, the levels of these compounds were slightly increased or not significantly changed in W7-1 at different days after planting. The level of ALA was significantly higher in W7-2. These data demonstrated that uncertain factors resulted in the reduction of Chl intermediates and led to abnormal leaf color changes. Accumulation of carbohydrate in leaves of the two phenotypes As shown in Fig. 9, the total soluble sugar and fructose content in the two varieties leaves were increased markedly during different growth periods. The levels of total soluble sugar and fructose in W7-2 were significantly higher than in W7-1 during different growth periods; these 12


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were 39.4% and 45.6% higher, respectively, than in W7-1 at 13 DAP. The level of sucrose also was observably increased in W7-1 but decreased dramatically from 29 to 37 DAP or had no significant changes during other growth periods for W7-2 (Fig. 9C). Additionally, the level of glucose was increased dramatically in both varieties from 13 DAP to 29 DAP, but there was no significant difference in W7-1 from 29 to 45 DAP. Glucose content continued to increase in W7-2 during the same periods (Fig. 9D). The starch content and the ratio of starch to total soluble sugar were measured; the starch content was significantly accumulated from 13 DAP to 21 DAP and declined markedly in the latter three periods in W7-2. The starch content showed a slightly increased in W7-1 from 13 to 29 DAP, and significantly decreased from 29 to 45 DAP, but the level of starch in W7-2 was significantly higher than in W7-1 during the different growth periods (Fig. 9E). The levels of starch and soluble sugar were further compared between the two varieties. The ratio of starch to soluble sugar increased, indicating that the starch was significantly accumulated in the early stages and was perhaps transformed to soluble sugar during the later stages in W7-2 (Fig. 9F). Meanwhile, the ratio decreased in W7-1; this may be because most of the energy produced by photosynthesis was fixed in the form of soluble sugar. Expression profiles of selected genes associated with chlorophyll metabolism and photosynthesis at two stages in the two genotypes To explore the underlying reasons for the DEPs between W7-2 and W7-1, the expression pattern of selected genes involved in chlorophyll biosynthesis, photosynthesis, and carbohydrate and energy metabolism were analyzed. In terms of chlorophyll metabolism, the expression of HEMA1 was significantly lower in W7-2 (Fig. 10A) There was no significant change in W7-1. CHLI1, CHLD and TRX3 were significantly increased in W7-1 and were down-regulated in W7-2 (Fig. 10B, D, G). However, the expression levels of CHLH and GUN4 were significantly decreased in W7-2 (Fig. 10C, F), and the expression levels of GUN4 was no significant difference in W7-1. The transcript expression levels of CHLI1, CHLH and CHLD showed respective 0.21-fold, 0.22-fold and 0.22-fold changes in W7-2, and these were 3.26fold, 0.19-fold and 5.31-fold in W7-1. The expression level of PORB was greater than at 21 DAP in W7-2 but in W7-1 was significantly lower than at 21 DAP. The mRNA levels of the photosynthesis-related proteins (psaA, psaC, LHCB5 and LHCB3) were measured. The proteins psaA, LHCB5 and LHCB3 showed significantly lower levels (Fig. 10H, J, K), except for psaC, 13



in W7-2. By comparison, the expression levels of the genes were significantly increased or no obvious changes except for LHCB3 at 29 DAP in W7-1. The expression levels of genes involved in carbohydrate and energy metabolism were measured (Fig. 10L-O). Malate dehydrogenase (MDH2), fructose-bisphosphate aldolase (FBA1) and Ribulose bisphosphate carboxylase (rbcL) showed lower levels in W7-2 except Phosphoglycerate kinase (PGK1) but were significantly higher or had no significant differences in W7-1. Discussion Proteins are the basis of life and are the specific practitioners of life activities. Proteomic research contributes to reveal complex color changes in wucai leaves, and this approach can provide new understanding about the wucai leaves’ color response to cold acclimation. In this study, an iTRAQ-based comparative proteomics analysis was made between W7-2 (yellow inner leaves) and W7-1 (green inner leaves) at two developmental stages (21 DAP and 29 DAP). Using bioinformatics methods, we identified many DEPs, including clear functional proteins and uncharacterized proteins (Tables S5 and S6). Although their bioinformation was analyzed, the function of these proteins could still be uncertain. Thus, several DEPs associated with leaf color are discussed in detail below. Based on proteomic data, we identified 77 up-regulated and 53 down-regulated proteins from W7-2 vs W7-1 at 21 DAP (Fig. 5). Meanwhile, there were 53 up-regulated and 35 down-regulated proteins from W7-2 vs W7-1 at 29 DAP. The proteomics comparative analysis of the two genotypes at two different developmental stages yielded insights into the mechanism of leaf color change in W7-2. Key differentially expressed proteins involved in chlorophyll biosynthesis Leaf is the main site of photosynthesis, which is helpful for plant growth and bioenergy synthesis.39,40 The chlorophyll biosynthesis can be divided into three parts, including the synthesis of ALA, Proto IX and the synthesis of Mg-Proto IX, Pchlide and Chlide.41 In our study, contents of Chl a, Chl b and total Chl in W7-2 were significantly lower than in W7-1 at different developmental stages (Fig. 3). Consistent with this observation, the key enzymes involved in Chl biosynthesis as well as the mRNA and protein levels were decreased in W7-2 compared with W7-1 at 21 DAP. In Arabidopsis, there are three differentially expressed POR isoenzymes, named PORA, PORB and PORC. Garrone42 found that the substrate binding affinity of PORB for Pchlide was five-fold higher than that of PORA, and the catalytic 14


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efficiency (kcat/Km) was about six-fold higher than that of PORA. But PORA and PORB have the same catalytic mechanism. In this study, only PORB was identified; the enzyme was downregulated in W7-2 compared with W7-1 at 21 DAP. This could be the reason why the chlorophyll level of W7-2 was significantly lower than that of W7-1. However, the transcript expression level of PORB was much higher at 29 DAP compared to 21 DAP in W7-2 (Fig. 10E), which could be associated with a significant decrease in Pchlide in W7-2 (Fig. 8F). In the present study, glutamyl-tRNA (Gln) amidotransferase subunit A protein was up-regulated in chloroplastic/mitochondria in W7-2 compared with W7-1 at 21 DAP. Glutamyl-tRNA synthetase, which belongs to the class I aminoacyl-tRNA synthetases, exhibits similarities to glutamyl-tRNA synthetase in structure and catalytic properties.43 The protein catalyzes Glu to ALA, as indicated by higher levels of ALA at 21 DAP in W7-2. Many studies have demonstrated that reduced transcript level of HEMA1 can attenuate the activity of GluTR and inhibits the biosynthesis of heme and chlorophyll.44,45 As shown in Fig. 10A, the expression of HEMA1 was down-regulated at 29 DAP compared to 21 DAP in W7-2, leading to constant decreasing of ALA content in W7-2 and hampering Chl biosynthesis. After the color change of W7-2 leaves, CHLI1 was up-regulated in W7-2 vs W7-1 at 29 DAP. The three subunits CHLH, CHLI and CHLD constitute the Mg-protoporphyrin IX chelatase in plants, among which CHLH is primarily responsible to catalyze insertion of Mg2+ into Proto IX in the presence of ATP and Mg2+.46 The results showed that the up-regulation of CHLI1, which led to Proto IX, was significantly decreased at 29 DAP in W7-2. Many reports have focused on the regulatory mechanisms governing Mg-chelatase activity. TRXs redox regulates the ATPase activity of the CHLI subunit.47,48 GUN4 is a porphyrin-binding protein, which can improve the efficiency of porphyrin-substrate transformation into metalloporphyrin products, and stimulate the activity of chelatase at the physiologically significant concentration of Mg2+.49,50 The TRX3 protein and uncharacterized protein (GUN4) were identified and upregulated in W7-2 vs W7-1 at 21 DAP. These proteins were down-regulated in W7-2, consistent with the expression levels of CHLI1 (Fig. 10). It is possible that down-regulation of these proteins resulted in decreases of Mg-chelatase activity. The result was the opposite of that in W7-1, where the expression level of CHLI1 was consistent with the that of TRX3 was significantly up-regulated, which may be the reason that W7-1 maintained the green phenotype. 15



In a previous study, a chlorophyll-deficient soybean mutant (cd1) displayed lower chlorophyll content and abnormal chloroplasts, and accompanied with a chlorina phenotype compared to WT. Map-based cloning showed that cd1 has a missense mutation (G1709A) in GmCHLI1b.51 In rice, the leaves of the ygl mutant seedling show yellow-green at 20°C and normal green at 34°C. The transcript level of the YGL was significantly reduced in the ygl mutant at 20°C, whereas this result was reversed at 34°C compared to the wild type plant. The YGL encodes a Mg2+-chelatase CHLD subunit that was a new allele of Chl1/YGL7 by genetic and molecular analysis.52 However, there are few reports concerning the transformation of a phenotype as in wucai in naturally decreasing temperatures. Key differentially expressed proteins involved in photosynthesis and carbohydrate metabolism Six light-harvesting complex CAB and two PSI proteins were down-regulated in W7-2 compared with W7-1 at 21 DAP (Table S5). This was consistent with the result of a decrease in the Chl content. (Fig. 3), affecting the presence of Chl molecules.53,54 The down-regulation of CAB and PORB could be the reason for promoting the reduction of Chl content in W7-2. These results were similar to previous studies indicating that changes in CAB on the membrane of the internal capsule were a crucial step in disrupting chlorophyll degradation.55 After the leaves of W7-2 changed color, CAB (chloroplastic) protein was still down-regulated. In the process of cold acclimation, plant cells accumulate soluble sugar to protect them from damage caused by cold stress. The sugars can serve as osmoprotectants and nutrient supplements, and they may interact with the lipid bilayer. In the present research, there were different increases in water-soluble sugar including the soluble sugar, fructose, glucose and sucrose contents from 13 to 45 DAP in both varieties (Fig. 9), indicating that plants began to accumulate carbohydrate to acclimate to the environment.56 Carbohydrate metabolism related proteins were found in table S5, such as FBA1 and PGK1 involved in glycolysis, as well as MDH2 involved in the TCA cycle. The abundances of these proteins were lower in W7-2 compared with W7-1 at 21 DAP. The changes in protein abundance were consistent with gene transcriptional levels in W7-2 (Fig. 10L-O). However, the fructose content of W7-2 was significantly higher and continuously increased compared to W7-1 (Fig. 9B), and the ratio of starch/total soluble sugar was dramatically decreased from 21 to 45 DAP (Fig. 9F). These 16


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results may be due to most of the starch being converted to soluble sugar as an osmoprotectant to help plants adapt to low temperatures.57 Soluble sugar might be transported from the green external leaves to the yellow inner leaves to maintain normal growth and development of the whole plant. Meanwhile, the degradation of chlorophyll led to a decrease in photosynthetic efficiency, resulting in a reduction in starch synthesis. The two major subtypes of FBA are present in plants, one in the cytoplasm and the other in the chloroplast,58 and the two forms have different characteristics59. As a part of sucrose biosynthesis pathway, the cytosol of FBA is a vital metabolic enzyme in glycolysis/gluconeogenesis pathway.60,61 This pathway can cause the synthesis and accumulation of energy substances.62 Liu63 found that rice mutant OsNOA1/RIF1 RNAi, its seedlings showed chlorosis, has a decreased pigment content and photosystem II efficiency. In this study, FBA1 protein abundance and the gene expression levels were down-regulated in W7-2 (Table S5, Fig. 10N), which would lead to an inability to accumulate large amounts of sucrose (Fig. 9C) and interfere with Chl biosynthesis.64 Further research is needed to determine whether carbohydrate metabolism regulates chlorophyll biosynthesis. In addition, the rbcL and UTP-glucose-1-phosphate uridylyl transferase (UDP1) were up-regulated in W7-2 vs W7-1 at 21 DAP. These enzymes participate in carbon fixation of photosynthesis and catalyze the Calvin cycle. Up-regulation of the two enzymes might promote leaf coloring in order to accumulate energy products to adapt to the cold environment. The UDP1 was up-regulated in W7-2 compared with W7-1 at 29 DAP, but the ribulose bisphosphate carboxylase large chain and ribulose-phosphate 3-epimerase proteins were downregulated, suggesting that photosynthesis was damaged in W7-2. Key differentially expressed proteins involved in ribosome biosynthesis and protein modification Ribosomal biogenesis is a key step in the growth and development of organisms. Therefore, the cell with rapidly developing distributes most of their transcriptional/translational capabilities to enhance the biosynthesis of rRNAs and ribosomal proteins.65,66 In eukaryotic cells, the synergistic synthesis of four ribosomal RNAs (rRNAs) and more than 70 ribosomal proteins promotes the ribosome biogenesis, and the cellular physiological state strictly regulates the transcription/translation of each component.67 In this study, the ribosomal protein L19, 30S ribosomal protein S18 (Chloroplastic), 40S ribosomal protein S3a, 40S ribosomal protein S12 17



and 60S ribosomal protein L29 proteins were identified and were up-regulated in W7-2 vs W7-1 at 21 DAP (Table S5). Ribosomal proteins have many functions in regulating cellular metabolism and protein synthesis. Chloroplast is one of the two semi-autonomous organelles in plant cells, the nuclear gene encodes more than 90% of the protein in the chloroplast.68 The up-regulation of these proteins could be great of significance to protein metabolism in the course of chloroplast biogenesis and development of defenses to cold accumulation. After the W7-2 leaves changed color, six ribosomal proteins were identified that were up-regulated in W7-2 vs W7-1 at 29 DAP (Table S6), and among the identified proteins, five were predicted to be present in the chloroplasts. The results showed that chloroplast protein metabolism was still relatively active. It is possible that this was an adaptation of cultivated plants to cold acclimation to maintain chloroplast protein metabolism. As shown in Table S6, there were two peptidyl-prolyl cis-trans isomerase proteins that accelerate the folding of proteins. These enzymes catalyze the cis-trans isomerization of proline amidic peptide bonds in oligopeptides. The up-regulation of peptidyl-prolyl cis-trans isomerase proteins may be closely related to the expression of ribosomal proteins in W7-2 vs W7-1 at 29 DAP. Uncharacterized proteins

According to bioinformatics comparison results, we identified many uncharacterized proteins (Table S5 and S6). These proteins identified possible genes by comparison. However, the bioinformatic analysis could not identify the roles of these proteins, and their functions remain unclear. Conclusion The 21 DAP and 29 DAP periods were critical stages for leaf color changes in W7-2. The Chl contents were decreased from 13 DAP to 45 DAP in W7-2, and this was consistent with the changes in color value. The comparative proteomics analysis by iTRAQ identified 77 upaccumulated proteins and 53 down-accumulated proteins at 21 DAP in W7-2 compared with W7-1 and 53 up-accumulated proteins and 35 down-accumulated proteins at 29 DAP. There were many pathways that are underlying related to chlorophyll deficiency, including chlorophyll biosynthesis, photosynthesis, carbon fixation and energy metabolism, ribosome metabolism and posttranslational modification. DEPs related to chlorophyll biosynthesis were 18


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identified as PORB and CHLI1; these proteins closely participated in the leaf color change of W7-2 as they contributed to reduced Chl content and reduced intermediates of chlorophyll metabolism. These results provide further insight into the metabolic mechanism of W7-2 leaf color change (Fig. 11). Further studies of the molecular mechanism were performed to examine the underlying mechanism of the regulation of chlorophyll metabolism involved in W7-2 leaf coloring.

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ASSOCIATED CONTENT Supporting information

Table S1: Simulate local weather change values. Table S2: Primers for fragment amplification of differentially expressed proteins related to genes. Table S3: Color values change on the different days after planting (DAP). R-G: Red-Green; Y-B: Yellow-Blue. Table S4: Identification and database search of differentially expressed proteins in common at 21 DAP and 29 DAP. Table S5: Identification and database search of differentially expressed proteins in W7-2 vs W7-1 at 21 DAP. Table S6: Identification and database search of differentially expressed protein in W7-2 vs W7-1 at 29 DAP. Three Supplementary File: iTRAQ results. (XLSX). A supplemental Figure file: Gene ontology (GO) annotation of the differentially expressed proteins at 21 DAP and 29 DAP in W7-2 vs W7-1. A supplemental material file: The leaf phenotype of W7-2 at room temperature. AUTHOR INFORMATION *Corresponding author: Lingyun Yuan and Chenggang Wang Lingyun Yuan Tel./Fax. +86 0551-65786212 E-mail: [email protected] Chenggang Wang Tel./Fax. +86 0551-65786212 E-mail: [email protected] Notes

The authors declare no competing financial interest. Acknowledgments

This work was funded by the National Natural Science Foundation of China (No. 31701910), the National Key R & D Program of China (2017YFD0101803) and the Major Science and Technology Projects of Anhui Province, China (17030701013).

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Author Contribution

Lingyun Yuan, Chenggang Wang and Shilei Xie designed the experiment. Shilei Xie wrote the manuscript. Shilei Xie, Yushan Zheng, Jie Wang and Mengru Zhao carried out the experiments. Shidong Zhu, Guohu Chen and Jinfeng Hou supervised the study and Libing Nie helped perform the experiments. All authors have read and approved the final manuscript. Mass spectrometry data The

mass

spectrometry

data

have

been

deposited

to

the

PRIDE

Archive

(http://www.ebi.ac.uk/pride/archive/) via the PRIDE partner repository with the data set identifier PXD013323 and 10.6019/PXD013323.

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(46) Richter, A.S.; Grimm. B. Thiol-based redox control of enzymes involved in the tetrapyrrole biosynthesis pathway in plants. Front. Plant Sci. 2013, 4, 371. (47) Ikegami, A; Yoshimura, N.; Motohashi, K.; Takahashi, S.; Romano, P.G.; Hisabori, T.; Takamiya, K.; Masuda, T. The CHLI1 subunit of Arabidopsis thaliana magnesium chelatase is a target protein of the chloroplast thioredoxin. J. Biol. Chem. 2007, 282, 1928–19291. (48) Luo, T.; Fan, T.; Liu, Y.; Rothbart, M.; Yu, J.; Zhou, S.; Grimm, B.; Luo, M. Thioredoxin redox regulates ATPase activity of magnesium chelatase CHLI subunit and modulates redox mediated signaling in tetrapyrrole biosynthesis and homeostasis of reactive oxygen species in pea plants. Plant Physiol. 2012, 159, 118–130. (49) Larkin, R.M.; Alonso, J.M.; Ecker, J.R.; Chory, J. GUN4, a regulator of chlorophyll synthesis and intracellular signaling. Science 2003, 299, 902-906. (50) Davison, P.A.; Schubert, H.L.; Reid, J.D.; Iorg, C.D.; Heroux, A.; Hill, C.P.; Hunter, C.N. Structural and biochemical characterization of Gun4 suggests a mechanism for its role in chlorophyll biosynthesis. Biochemistry 2005, 44, 7603–7612. (51) Du, H.; Qi, M.; Cui, X.; Cui, Y.; Yang, H.; Zhang, J.; Ma, Y.; Zhang, S.; Zhang, X. Proteomic and functional analysis of soybean chlorophyll-deficient mutant cd1 and the underlying gene encoding the CHLI subunit of Mg-chelatase. Molecular Breeding 2018, 38, 71. (52) Ruan, B.; Gao, Z.; Zhao, J.; Zhang, B.; Zhang, A.; Hong, K.; Qian, Q. The rice YGL gene encoding an Mg2+-chelatase ChlD subunit is affected by temperature for chlorophyll biosynthesis. Journal of Plant Biology 2017, 60(4), 314–321. (53) Horie, Y.; Ito, H.; Kusaba, M.; Tanaka, R.; Tanaka, A.; Participation of chlorophyll b reductase in the initial step of the degradation of light-harvesting chlorophyll a/b protein complexes in Arabidopsis. J. Biol. Chem. 2009, 284, 17449–17456. (54) Hortensteiner, S. Stay-green regulates chlorophyll and chlorophyll-binding protein degradation during senescence. Trends. Plant Sci. 2009, 14, 155–162. (55) Barry, C.S. The stay-green revolution: recent progress in deciphering the meciianisn is of chlorophyll degradation in higher plants. Plant Sci. 2009, 176, 325–333. (56) Ma, Y.; Zhang, Y.; Lu, J.; Shao, H. Roles of plant soluble sugars and their responses to plant cold stress. Afr. J. Biotechnol. 2009, 8, 2004–2010. 26


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(57) Maruyama, K.; Takeda, M.; Kidokoro, S.; Yamada, K.; Sakuma, Y.; Urano, K.; Fujita, M.; Yoshiwara, K.; Matsukura, S.; Morishita, Y.; Sasaki, R.; Suzuki, H.; Saito, K.; Shibata, D.; Shinozaki, K.; Yamaguchi-Shinozaki, K. Metabolic Pathways Involved in Cold Acclimation Identified by Integrated Analysis of Metabolites and Transcripts Regulated by DREB1A and DREB2A. PLANT PHYSIOLOGY 2009, 150(4), 1972–1980. (58) Gross, W.; Lenze, D.; Nowitzki, U.; Weiske, J.; Schnarrenberger, C. Characterization, cloning, and evolutionary history of the chloroplast and cytosolic class I aldolases of the red alga Galdieria sulphuraria. Gene 1999, 230(1), 7–14. (59) Li, H.M.; Chiu, C.C. Protein transport into chloroplasts. Annu. Rev. Plant. Biol. 2010, 61, 157–180. (60) Hu, J.; Aguirre, M.; Peto, C. A role for peroxisomes in photomorphogenesis and development of Arabidopsis. Science 2002, 297, 405–409. (61) Sapir-Mir, M.; Mett, A.; Belausov, E.; Tal-Meshulam, S.; Frydman, A.; Gidoni, D.; Eyal, Y. Peroxisomal localization of Arabidopsis isopentenyl diphosphate isomerases suggests that part of the plant isoprenoid mevalonic acid pathway is compartmentalized to peroxisomes, Plant Physio. 2008, 148, 1219–1228. (62) Konishi, H.; Yamane, H.; Maeshima, M.; Komatsu, S. Characterization of fructosebisphosphate aldolase regulated by gibberellin in roots of rice seedling. Plant Molecular Biology 2004, 56(6), 839–848. (63) Liu, H.; Lau, E.; Lam, M. P. Y.; Chu, H.; Li, S.; Huang, G.; Guo, P.; Wang, Ji.; Jiang, L.; Chu, I.; Lo, Clive.; Tao, Y. OsNOA1/RIF1 is a functional homolog of AtNOA1/RIF1: implication for a highly conserved plant cGTPase essential for chloroplast function. New Phytologis 2010, 187(1), 83–105. (64) Zhang, F.; Zhang, P.; Zhang, Y.; Wang S.C.; Qu L.H.; Liu, X.Q.; Luo, J. Identification of a peroxisomal-targeted aldolase involved in chlorophyll biosynthesis and sugar metabolism in rice. Plant Science 2016, 250, 205–215. (65) Moss, T.; Langlois, F.; Gagnon-Kugler, T.; Stefanovsky, V. A housekeeper with power of attorney: the rRNA genes in ribosome biogenesis. Cell. Mol. Life Sci. 2007, 64, 29–49. (66) Lempinen, H.; Shore, D. Growth control and ribosome biogenesis. Curr. Opin. Cell. Biol. 2009, 21, 855–863. 27



(67) Mayer, C.; Grummt, I. Ribosome biogenesis and cell growth: mTOR coordinates transcription by all three classes of nuclear RNA polymerases. Oncogene 2006, 25, 6384–6391. (68) Li, H.; Chiu, C.C. Protein Transport into Chloroplasts. Annual Review of Plant Biology 2010, 61(1), 157–180.

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Page 29 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For TOC Only

Photograph courtesy of Shilei Xie. Copyright 2018.

29



Figures

Photograph courtesy of Shilei Xie. Copyright 2018.

Fig. 1 A: Appearance of W7-1 and W7-2; Vertical cutting view about aerial part of W7-2. S1-S4: Four different samples. B: Sequence numbering of the heading leaves of W7-2. The P10-P19 is the edible inner leaf of W7-2. C: The two phenotypes at different days after planting. The scale bar is 10 cm. Position of leaves: the third fully expanded young leaves from the center.

30


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Page 31 of 38

A

W7-2

60

B

W7-1

W7-1

70

H* value

C* value

W7-2

80

45 30

60

15

50

0

40

5 9 13 17 21 25 29 33 37 41 45 49 53 57 DAP

5 9 13 17 21 25 29 33 37 41 45 49 53 57 DAP

Fig. 2 The change of Chroma (A) and Hue angle (B) value of the two varieties at different days after planting. Error bars

4

c

f

g

h

1

b

d

e

3 2

W7-2 W7-1

a

i j

13

21

29

37

DAP

45

C a

0.5

0.0

b

b

c

13

cd

21

b

de 29

de 37

W7-2 W7-1

3

a

a 2

1

0

b

c d

e

13

b

ef

21

f

29

4

g 37

3

DAP

45

W7-2 W7-1

a

a

b

1.0

B

D

W7-2 W7-1

1.5

The ratio of Chl a:b

0

Chlorophyll a content (mgg FW)

A

-1

Total Chlorophyll content (mgg-1 FW)

represent SD (± SD), and representative data from five independent experiments are presented.

Chlorophyll b content (mgg-1 FW)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ab

cd

bc e

cde

de

e

de

2 1

e 45

0

DAP

13

21

29

37

45

DAP

Fig. 3 The comparison of pigment contents in two varieties at different days after planting. (A) The level of total Chl at different days after planting. (B), (C) The levels of Chl a, Chl b at different days after planting. (D) Chl a/b ratio calculated from (B, C). Three individual plants of each cultivar were quantified, and the pigments were measured three times. Error bars represent SD (± SD). Different letters indicated statistically significant differences at the level of P < 0.05.

31



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Fig. 4 Basic information about identified protein by iTRAQ. (A) Proteome identification in wucai leaf; (B) The amount of proteins in the identified peptide; (C) The amount of proteins by the identified peptides.

32


Page 33 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


A

B

110

20

W7-2 vs W7-1 (21 DAP)

68

W7-2 vs W7-1 (29 DAP)

Fig. 5 Quantitative analysis of the proteome (A) and Venn analysis (B) between W7-2 and W7-1 at two growth stages; W7-2 vs W71 (21 DAP) stand for W7-2 compared to W7-1 at 21 days after planting; W7-2 vs W7-1 (29 DAP) stand for W7-2 compared to W7-1 at 29 days after planting.

33



Page 34 of 38

Fig. 6 The heatmap for the DEPs involving metabolic pathways. In the heatmap, the green color represents down-regulated DEPs and red color represents up-regulated DEPs.

B

A

Fig. 7 The significant entries according to the KEGG pathway enrichment analysis of differentially expressed proteins at (A) W7-2 vs W7-1 (21 DAP) and (B) W7-2 vs W7-1 (29 DAP).

34


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A

e

13

21

e

e

10 0

29

37

W 7-2 W 7-1

d

a

b

c

b

cd e

f

f

g

0.005

13

21

29

37

E 0.8

a

a

DAP

45

a

a

b

c d

e

0.2 0.0

e

13

e

e

10

c

21

29

e

d f

37

45

13

21

29

f

g

37

45

a

b

b

b

b

1.0 c

c d

0.5

0.0

13

21

e

29

f

37

45

0.4

DAP

W 7-2 W 7-1

0.6

ab

bc

a

bc

c

d e

0.2 0.0

DAP

DAP

W 7-2 W 7-1

1.5

0.8

0.6 0.4

c

20

F

W 7-2 W 7-1

Pchl content (gg-1 FW)

0.000

b

0

DAP

45

a 30

D

C

0.015

PBG content (gg-1 FW)

ALA content (gg-1 FW)

c d

0.010

b

ab

b

W 7-2 W 7-1

40

Proto IX content (gg-1 FW)

Glu content (molg-1 FW)

a

ab

30 20

B

W 7-2 W 7-1

40

Mg-Proto IX content (gg-1 FW)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


13

21

f

g

29

37

h 45

DAP

Fig. 8 The concentration of Chl synthesis intermediates in two varieties at different days after planting, the concentration of Glutamic (A), 5-Aminolevulinate (B), Porphobilinogen (C), Protoporphyrin IX (D) and Mg-protoporphyrin IX (E), Protochlorophyllide (F). Three individual plants of each cultivar were quantified. Each data point represents the mean (± SD) from three separate experiments. Different letters within a column indicate significant differences at P < 0.05.

35


a

15

bc

13

21

29

37

Sucrose content (mgg-1)

a

b

2

cd

cd c

de h

1 0

ef

f

g

13

21

29

37

45 DAP

E

30

W 7-2 W 7-1

a

25 20

b c

15

f

ef

de

d

cd ef

10

g

5 0

13

21

29

37

45 DAP

9 6

b

c e g

d

de

f h

3

i

13

21

29

45 DAP

37

D

W 7-2 W 7-1

3

a

0

45 DAP

W 7-2 W 7-1

6

Glucose content (mgg-1)

C

e

g

h

5 0

f

g

W 7-2 W 7-1

12

a b

4

2

0

the ratio of starch to total soluble sugar

10

b

c

d

Page 36 of 38

B

W 7-2 W 7-1

Fructose content (mgg-1)

A

20

4

Starch content (mgg-1)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Total soluble sugar content (mgg-1)


F

e

f

13

e

d

21

c

c

29

c

37

45 DAP W 7-2 W 7-1

4

b

3

a

c 2

e

d h

1 0

c

13

21

29

g gh

37

h h

45 DAP

Fig. 9 The level of soluble sugar and starch in two varieties at different days after planting. The amounts of total soluble sugar (A), fructose (B), sucrose (C), glucose (D) and starch (E) are shown. Each data point represents the mean (± SD) from three separate experiments. Different letters within a column indicate significant differences at P < 0.05.

36


21 DAP 29 DAP

GUN4

1.0

0.5

**

0.0

K 1.5

1.0

0.5

** ** W7-1

21 DAP 29 DAP

TRX3

1

** W7-1

* 4 3 2 1

W7-1

W7-2

**

4

2

**

0

*

W7-1

W7-2 21 DAP 29 DAP

psaA

4 3 2 1

* W7-1

I

psaC

*

5

*

0 W7-1

*

2.0 1.5 1.0 0.5

* W7-2

1.5

21 DAP 29 DAP

LHCB5

1.0

0.5

** 0.0

W7-1

W7-1

W7-2

N 1.5

21 DAP 29 DAP

FBA

1.0

0.5

*

O

3

**

W7-2 21 DAP 29 DAP

rbcL

2

1

* 0

0.0

W7-2

J

21 DAP 29 DAP

1

W7-1 21 DAP 29 DAP

**

2

0

0.0

21 DAP 29 DAP

MDH2

PORB

W7-2

2.5

W7-2

M 15

10

6

E3 Relative expression level

**

0

21 DAP 29 DAP

PGK1

0 W7-2

H5

W7-2

L5

*

W7-1

0

21 DAP 29 DAP

LHCB3

*

0.5

21 DAP 29 DAP

CHLD

0.0

W7-2

2

W7-2

Relative expression level

W7-1

G3

1.0

D8


F 1.5

21 DAP 29 DAP

CHLH


** W7-1

1.5


1

W7-2


W7-1


2

0

0.0

0.0

3

C


**

21 DAP 29 DAP

CHLI1


0.5

**



1.0

B4


21 DAP 29 DAP

HEMA1


A 1.5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60



Page 37 of 38

W7-1

W7-2

W7-1

W7-2

Fig. 10 qRT-PCR expression analysis of genes. (A, B, ..., G) Real-time RT-PCR analysis of genes involved in Chl biosynthesis in W7-2 and W7-1, respectively; (H, …, K) The genes involved in photosynthesis; (L, …, O) The genes involved in carbohydrate metabolism. Total RNA was extracted from newer-leaf of two varieties at 21 and 29 DAP. Data are means ± SD of three replicates. * indicates significant differences at P < 0.05 and ** indicate significant differences at P < 0.01. K

37



Page 38 of 38

Fig.11 Related metabolic pathways and regulation chlorophyll biosynthesis metabolic pathway of W7-2 and W7-1.

38


Comparative proteomic analysis reveals that chlorophyll metabolism

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