Proteomic profiling for metabolic pathways involved in interactive

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Proteomic profiling for metabolic pathways involved in interactive effects of elevated carbon dioxide and nitrogen on leaf growth in a perennial grass species Jingjin Yu, Ningli Fan, ran li, Lili Zhuang, qian xu, and Bingru Huang J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00951 • Publication Date (Web): 13 May 2019 Downloaded from http://pubs.acs.org on May 15, 2019

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Journal of Proteome Research

Proteomic profiling for metabolic pathways involved in interactive effects of elevated carbon dioxide and nitrogen on leaf growth in a perennial grass species

Jingjin Yua, Ningli Fana, Ran Li a, Lili Zhuang a, Qian Xua*, Bingru Huangb * a.College

of Agro-grassland Science, Nanjing Agricultural University; Nanjing,

210095, P.R. China b.Department

of Plant Biology and Pathology; Rutgers, the State University of New

Jersey, New Brunswick, NJ, 08901, USA

*

Corresponding author: Qian Xu and Bingru Huang

E-mail: [email protected]; [email protected]

1

ACS Paragon Plus Environment

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ABSTRACT Elevated atmospheric CO2 and nitrogen are major environmental factors affecting shoot growth. The objectives of this study are to determine the interactive effects of elevated CO2 and nitrogen on leaf growth in tall fescue (Festuca arundinacea) and to identify major proteins and associated metabolic pathways underlying CO2-regulation of leaf growth under insufficient and sufficient nitrate conditions using proteomic analysis. Plants of tall fescue treated with low nitrate level (0.25 mM, LN), moderate nitrate level (4 mM, MN) and high nitrate level (32 mM, HN) were exposed to ambient (400 µmol mol-1) and elevated (800 µmol mol-1) CO2 concentrations in environment-controlled growth chambers. Increased atmospheric CO2 concentration increased leaf length and shoot biomass, which corresponded to increased content of indo-acetic acid, gibberellic acid, cytokinins and reduced content of abscisic acid under sufficient nitrate conditions (MN and HN conditions). Low nitrate supply limited shoot growth and hormonal responses to elevated CO2. Proteomic analysis of plants exposed to elevated CO2 under LN and MN conditions demonstrated the increases in the abundance of many proteins due to elevated CO2 under MN condition involved with cell cycle and proliferation, transcription and translation, photosynthesis (ribosomal and chlorophyll a/b-binding proteins), amino acids synthesis, sucrose and starch metabolism, as well as ABA signaling pathways (ABA-induced proteins). Our results revealed major proteins and associated metabolic pathways associated with the interactive effects of elevated CO2 and nitrate regulating leaf growth in a perennial grass species. Key words: elevated CO2; grass; hormone; leaf growth; nitrate; protein

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ABBREVIATIONS DEPs

differentially expressed proteins

GA

gibberellic acid

IAA

indole-3-acetic acid

ABA

abscisic acid

iPA

isopentenyl adenosine

LN

low nitrate

MN

moderate nitrate

HN

high nitrate

aCO2

ambient CO2

eCO2

elevated CO2

GO

gene ontology

3



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INTRODUCTION Atmospheric CO2 concentration has been increasing since the beginning of the industrial era, and is expected to double by the end of the twenty-first century.1 Elevated CO2 affects various aspects of plant growth and development, including leaf elongation.2,3

Elevated

CO2

also

affects

nitrate

assimilation

involving

photorespiration.4 Plant responses to elevated CO2 are affected by nutrient availability, including nitrogen.2,5 However, inconsistent conclusions have been made regarding the interaction of elevated CO2 and nitrogen availability. Several studies reported that nitrogen starvation limits the positive effects of CO2 on plant growth6,7 whereas others found that elevated CO2 enhanced biomass production under low nitrogen supply (0.25 mM NO3- ) but had no effects with high nitrogen supply (2.5 mM NO3-) in durum wheat.8 While it is generally found that elevated CO2 promotes leaf elongation in different plant species, the interaction with nitrogen has not been well documented. The mechanisms underlying the interaction between nitrogen and CO2 regulating leaf growth and elongation are not well understood. Leaf elongation is governed by cell division and cell expansion. The promotion of leaf elongation by elevated CO2 in dicotyledon, such as poplar (Populus alba), was attributed to both increased leaf cell expansion and division.9 However, while cell length was not influenced by elevated CO2 in leaves of wheat (Triticum aestivum), cell division rate was significantly higher in plants exposed to 900 ppm CO2 than those under 350 ppm CO2.10 Plant hormones play crucial roles in regulating leaf elongation governed by cell division and elongation.11 Gibberellic acid (GA) and indole-3-acetic acid (IAA) are known as promoters for cell elongation and proliferation in plants.12,13,14 Cytokinin mainly regulates cell division.15 Abscisic acid (ABA) is known as a growth inhibitor.16 Previous studies that examined the gene expression involved in the synthesis and degradation of hormones under different CO2 concentrations found that auxin conjugation and cytokinin glucosylation and degradation, as well as ABA synthesis were inhibited in the vascular cambium zone of aspen (P. tremuloides) by 4


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elevated CO2.17 It can be postulated that interactive effects of elevated CO2 and nitrate on leaf elongation may involve changes in hormone production that regulate cell division and elongation; however, major hormones involved are unclear. Proteins play key roles in regulating cellular growth and leaf elongation, including those directly involved in cell expansion, such as cell-wall loosening proteins,18,19 proteins regulating cell division, such as cyclins20, and metabolic proteins catalyzing hormone synthesis and other numerous metabolic processes.21,22 Enhanced leaf elongation by elevated CO2 and interaction with nitrate may be associated with the alteration of leaf proteome. However, proteins and associated metabolic pathways underlying CO2 regulation of leaf growth under different nitrate conditions were not clear. Therefore, the objectives of this study are to determine the interactive effects of elevated CO2 and nitrate on leaf growth in tall fescue (Festuca arundinacea), a widely used forage and turf grass and to identify major proteins and associated metabolic processes underlying CO2-regulation of leaf growth under insufficient and sufficient nitrate conditions using proteomic analysis. MATERIALS AND METHODS Plant materials and treatments Seedlings of tall fescue cultivar ‘Barlexas’ were established from seeds in fritted clay for two weeks (2017.2.10-2017.2.14) and transferred into plastic bins (40×30×14 cm) filled with modified Hoagland solution.23 The plants were maintained in a walk-in growth chamber controlled at 24/20 ℃

(day/night) temperature, 60% relative

humidity, and 14-h photoperiod with photosynthetically-active radiation of 680 μmol m-2s-1 at the canopy level. Ten-week old seedlings of uniform sizes were transferred to 24 plastic containers (20 cm in diameter and 22 cm in depth) filled with half-strength Hoagland’s nutrient solution with three concentrations of nitrate, 0.25 mM (low nitrate, LN), 4 mM (moderate nitrate, MN) and 32 mM (high nitrate, HN). All other nutrient elements were maintained at the sufficient rates as in the typical half-strength 5



Hoagland’s nutrient solution. Plants were treated with three different rates of nitrate for 40 d (2017.4.13-2017.5.23) in the walk-in growth chamber under the same condition as described above. Each nitrate treatment was repeated in four containers as four replicates and there were 25 plants in each container. The nutrient solution was changed every week and aerated constantly by air pumps (115 V, 60 Hz, Tetra Blacksburg, VA). Plants treated with three nitrate treatments were immediately exposed to ambient CO2 (aCO2) concentration at 400±10 µmol mol-1 or elevated CO2 (eCO2) concentration at 800±10 µmol mol-1. Plants were exposed to CO2 treatments for 40 d (2017.4.13-2017.5.23) and 24 hours a day. The control systems of CO2 concentration in growth chambers were described in Yu et al. (2014).24 The experiment was conducted in a split plot design with aCO2 and eCO2 concentration as the main plots and different concentrations of nitrate as the subplots with each treatment being repeated in four replicates. Each CO2 treatment was repeated in two chambers simultaneously and plants in two CO2 treatments with different nitrate levels were relocated weekly across four chambers to avoid potential spatial environmental variations among chambers. Evaluation of shoot height, shoot biomass and cell length and number Leaf length was measured from the base to the tip of individual elongating leaves (first fully expanded leaf in each plant) using a ruler. Shoot biomass was measured after shoots including leaves and stems were dried in an oven at 80 ℃ for 5 days until the weight was no longer changed. At the end of the treatment, 10 of the first fully-expanded leaves in main stem were cut at the base and transferred into methanol immediately for chlorophyll removal. Then the leaves were transferred to 85% lactic acid for storage. The abaxial surface of leaf was brushed by nail polish and a fine transparent negative film of the epidermis was obtained. The picture of epidermis was observed under microscope and captured by a camera. The length of elongated cells was measured by software 6


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Digimizer (MedCalc Software bvba, Ostend, Belgium) and compared among different treatments. The characteristics of each type of epidermal cells were according to the previous report of Botwright25 in regard to wheat leaves. The interstomatal cells are the subsidiary cells between two stomata and elongated cells are long, unspecialized cells between the two cell rows of interstomatal and guard cells. Quantification of endogenous phytohormone content The 5 cm basal segments of the elongating leaves were harvested to determine the endogenous phytohormones using enzyme-linked immunosorbent assay (ELISA), including iPA (isopentenyl adenosine), IAA, ABA, and GA3 (the phytohomone assay kit, Beinong tianyi biological Co. Ltd., China). A total of 0.2 g of leaves were homogenized and extracted in 3 mL of 80% methyl alcohol including 1mM BHT (Tert-butyl p-methylpheno) for 4 h and then centrifuged for 8 min at 4 °C. The supernatant was collected and separated using a C18 column with a mobile phase of 80% methyl alcohol. The extraction was concentrated by vacuum drying and dissolved in 2 mL PBS solution (pH 7.5, with 0.1% Tween-20 and 1% gelatin). For the ELISA assay, 50 μL of sample dilution and standard hormone solution were added in 96-cell microtiter plate and then 50 μL of antibody dilution. After that, the plate was incubated for 30 min at 37 °C. The solution in plate was discarded, and each plate well was washed with 200 μL washing buffer four times. After the plate was dried, 50 μL of second antibody was added to each plate well, and the plate was incubated for 30 min at 37 °C. After the plate wells were washed four times with washing buffer, the o-phenylenediamine solution were added in each well. Terminating the reaction with 20μL sulfuric acid solution (2 mM) when the color of each cell could be distinguished. The absorbance of reaction mixture was read at 490 nm on a microplate reader (Synergy HTX, Bio Tek, USA). Protein Extraction and iTRAQ Labeling Three biological replicates were used to perform the proteome analysis. The 5-cm basal segments of the elongating leaves were used to extract the proteins for the 7



iTRAQ assay. For each replicate, 0.1 g frozen plant tissue was ground and the fine powder was extracted in 1 mL phenol extraction buffer for 10 min at room temperature. 1 mL phenol saturated with Tris-HCl (pH 8.0) was added to the samples and the mixture was shaking for 40 min at 4°C. After centrifuging at 5000 g for 15 min at 4°C, the upper phenolic phase was collected. Cold 0.1 M ammonium acetate methanol solution (five volumes of the collected phenolic phase) was added and incubated at -20°C for 12 h. The sediment was collected after centrifuging the sample at 12000 g for 10 min at 4°C and was mixed up with five volumes of cold methanol for washing. The washing step was repeated one more time, and the pellet was washed twice with five volumes of cold acetone. Then the pellet was dried at room temperature for 2 min and re-suspended in 300 μL lysate solution at room temperature for 3 h. Then the solution was centrifuged at 12000 g for 10 min at room temperature, and the supernatant was the extracted protein solution. The concentrations of the protein extracts were determined by the BCA method26. The sample was stored at -70°C. The protein was digested by trypsin based on the method of filter-aided sample preparation (FASP)27. Up to 100 μg protein extract was mixed with 120 μL reducing buffer (10 mM dithiothreitol, 8 M urea, 100 mM tetraethylammonium bromide (TEAB), pH 8.0) and incubated at 60°C for 1 h. The reaction solution in the 10K ultrafiltration tube was added iodoacetamide to reach the final concentration of 50 mM and incubated in the dark at room temperature for 40 min. The filter units were centrifuged at 12000 rpm for 20 min and the flow-through in the collection tube was discarded. The filter units were washed by 100 μL 100 mM TEAB twice and then transferred to new collection tubes. 100 μL 100 mM TEAB and 2 μL sequencing-grade trypsin (1 μg μL-1) was added to the units and incubated at 37°C for 12 h. The peptide solution was collected by centrifugation at 12000 rpm for 20 min and was lyophilized. The sample was reconstituted in 100 μL 100 mM TEAB and then 40 μL sample was transferred to a new tube for labeling. Each required vial of iTRAQ reagent was equilibrated to room temperature and added 200 μL of 8


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isopropanol. Each vial was vortexed to mix and spin and the step was repeated one more time. 100 μL iTRAQ reagent was transferred to the sample tube and incubated at room temperature for 2 h. 200 µL water was added to quench the labeling reaction. The solution was lyophilized and stored at -70°C. The iTRAQ reagent was labelled to the samples as the following scheme: the LN with ambient CO2 sample was labelled as 113, the MN with ambient CO2 sample was labelled as 114, the LN with elevated CO2 sample was labelled as 115, the MN with elevated CO2 sample was labelled as 116. Every treatment combination was replicated three times. LC−ESI−MS/MS Analysis All analyses were performed by a Triple TOF 5600 mass spectrometer (SCIEX, USA) equipped with a Nanospray III source (SCIEX, USA). Samples were loaded by a capillary C18 trap column (3 cm × 100 µm) and then separated by a C18 column (15 cm × 75 µm) on an Eksigent nanoLC-1D plus system (SCIEX, USA). The flow rate was 300 nL/min and linear gradient was 90 min (from 5 – 85% B over 67 min; mobile phase A = 2% ACN/0.1% FA and B = 95% ACN/0.1% FA). Data were acquired with a 2.4 kV ion spray voltage, 35 psi curtain gas, 5 psi nebulizer gas, and an interface heater temperature of 150°C. The MS scanned between 400 and 1500 with an accumulation time of 250 ms. For IDA, 30 MS/MS spectra (80 ms each, mass range 100 – 1500) of MS peaks above intensity 260 and having a charge state of between 2 and 5 were acquired. A rolling collision energy voltage was used for CID fragmentation for MS/MS spectra acquisitions. Mass was dynamically excluded for 22 seconds. Database Search and Protein Quantification ProteinPilot software (v.5.0) was used to search all of the Triple TOF 5600 MS/MS raw data thoroughly against the sample protein database translated from a cDNA library kept by our lab, which included 187681 cDNA sequences. Database searching was performed with trypsin digestion specificity and cysteine alkylation. 9



The peptide mass tolerance was 20 ppm and fragment mass tolerance was 0.1Da. The fixed modification was Carbamidomethyl (C), iTRAQ8plex (N-term), iTRAQ8plex (K) and variable modification was Oxidation (M). For protein quantification method, iTRAQ-8-plex was selected. The protein quantification strategy is mainly based on Paragon™ Algorithm integrated in the ProteinPilot software. The raw MS data was automatically processed using ProteinPilot according to manufactures instructions. The raw peptide identification results from Paragon™ Algorithm searches were further processed by the Pro Group™ Algorithm to determine the minimal set of proteins that can be reported for a given protein confidence threshold. In each group, one protein is designated the representative winner protein. A global false discovery rate (FDR) of < 1% was used and peptide groups considered for quantification required at least 2 peptides. Only peptides with unused score (≥1.3) at the 95% confidence interval were identified as counted confident proteins to reduce the probability of false peptide identification, and at least one unique peptide was involved in the identification of every confident protein. The low confidence, auto-shared peptides are excluded for quantification. To obtain a comprehensive observation of sample proteins, comparative proteomic analysis was evaluated using v-lookup function from three biological replicates. Only proteins that contained at least one peptide and that were detected in all three replicates were used in quantification. Those proteins with missing quantification values will not be considered for further analysis. Proteins with a p-value (T-test) below 0.05 were considered as statistically significant. For the all quantified proteins, only those showing a fold change of above 1.2 or below 1/1.2 (for the mean of the three replicates, and P < 0.05) in the quantitative ratios were considered as DEPs (differentially expressed proteins). All ratios (both the average ratio for proteins and the individual peptide ratios) can be corrected for bias. During bias correction, the software identifies the median average protein ratio and corrects it to unity, and then applies this factor to all quantitation results.Functional annotations of proteins were conducted using OmicsBean software and Blast2GO program against 10


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the non-redundant protein database (NR; NCBI; https://www.blast2go.com/). The KEGG database (http://www.genome.jp/kegg/) were used to classify and group these identified proteins. Statistical Analysis Data were analyzed using SPSS 18.0 (SPSS Inc., Chicago, IL, USA). Analysis of variance (ANOVA) was used to determine differences among different treatments after data normality and equal variance analysis. The means ± SE were calculated for each parameter. Means were tested with least significant difference (LSD) with P ≤ 0.05. RESULTS Interactive effects of elevated CO2 and nitrate rate on leaf growth and shoot biomass Elevated CO2 significantly enhanced leaf length and shoot biomass of tall fescue supplied with sufficient nitrate (MN with 4 mM and HN with 32 mM NO3-), but had no effects on leaf and shoot growth under low nitrate conditions (LN with 0.25 mM NO3-) (Figure 1A). Leaf length and biomass were increased due to elevated CO2 by 36% and 25% under MN condition, respectively, while under HN condition leaf length and shoot biomass increased by 23% and 20%, respectively (Figure 1B and C). Elevated CO2 had no effects on leaf cell length regardless of N concentrations, but significantly increased leaf cell number under both MN and HN conditions (Figure 2). Neither cell length nor cell number of the leaf was affected by elevated CO2 under LN condition. Interactive effects of elevated CO2 and nitrate rate on endogenous hormone content in leaves No significant effects of elevated CO2 were detected on the content of IAA, GA, iPA and ABA under LN conditions. Under MN and HN conditions, elevated CO2 11



significantly enhanced the content of IAA, GA and iPA (Figure 3A, B, C). Plants exposed to elevated CO2 had 100% and 200% higher iPA content than that at ambient CO2 under MN and HN conditions, respectively, while the corresponding increases were 90% and 62% for IAA and 133% and 117% for GA, respectively. The content of ABA decreased by 33% and 35% in MN and HN conditions, respectively, due to elevated CO2 treatment, but was not affected by elevated atmospheric CO2 in LN treatment. Interactive effects of elevated CO2 and nitrate rate on proteomic profiles In order to examine the metabolic pathways involved in the differential effects of elevated CO2 on the growth of tall fescue under different rates of nitrate, proteomic profiling was performed for plants grown under LN and MN conditions exposed to aCO2 or eCO2. A total of 326 proteins exhibited differential responses to ambient and elevated CO2, with 84 up-regulated and 102 down-regulated by elevated CO2 treatment under LN conditions, 65 up-regulated and 75 down-regulated by elevated CO2 under MN conditions. There were 31 DEPs up-regulated and 41 DEPs down-regulated by elevated CO2 under both LN and MN conditions, respectively (Figure 4). A GO (Gene Ontology) category enrichment analysis indicated that the 326 identified DEPs responsive to elevated CO2 were enriched in various biological processes (Figure 5A), molecular functions (Figure 5B), and cellular components (Figure 5C). For the biological process, most of the DEPs response to the elevated CO2 were enriched in metabolic process, single-organism process, and cellular process. There were more up-regulated DEPs induced by the elevated CO2 in the MN condition than in the LN condition among most biological processes except metabolic process, single-organism process, response to stimulus, and negative regulation of biological process, while the number of down-regulated DEPs due to the elevated CO2 were more in LN condition than in MN condition in all the biological processes (Figure 5A). 12


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Based on the molecular function classification, the proteins affected by elevated CO2 included binding proteins, oxidoreductase, transferase, hydrolase, peroxidase and cofactor. There were more DEPs down-regulated by elevated CO2 in LN condition than MN condition in most categories of molecular function, except transferase activity and hydrolase activity. In the up-regulated proteins induced by elevated CO2, more DEPs were enriched in MN condition than in LN condition in metal cluster binding, carbohydrate derivative binding, small molecule binding and lysase activity (Figure 5A). Most of the DEPs were located in the chloroplast, nucleus and ribosome according to the cellular component analysis (Figure 5C). The DEPs located in different cellular components were regulated differently by elevated CO2 in different N supplies. Under the LN condition, most of the DEPs located in the chloroplast, nucleus and ribosome were down-regulated by high CO2 concentration. Under MN condition, however, 10 DEPs were up-regulated and 4 DEPs were down-regulated by elevated CO2 in the nucleus, while 20 DEPs were up-regulated and 20 DEPs were down-regulated in the chloroplast. Metabolic Pathways associated with the interactive effects of elevated CO2 and nitrate rate Proteins involved in photosynthesis and carbohydrate metabolism or catabolism exhibited differential responses to elevated CO2 under LN and MN conditions. As shown in the Figure 6, the protein abundance of enzymes in the chlorophyll a synthesis pathway, including CHLH (Magnesium-chelatase subunit H), PORA (Protochlorophyllide reductase A), and CHLP (Geranylgeranyl diphosphate reductase) were increased by elevated CO2 under MN conditions, but not affected under LN conditions. The majority of DEPs related to the light harvesting process of photosynthesis were decreased or not affected by elevated CO2 under LN conditions, but increased under MN conditions, including three PSI reaction center proteins (PsaA, PsaD, and PsaN) and two antenna proteins (CAB1R, Chlorophyll a-b binding protein 1 and LHC1). Five PSII reaction center proteins, including PsbA, PsbB, PsbC, 13



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PsbD, and PsbH were down-regulated by elevated CO2 under LN conditions but not affected under MN conditions. The protein abundance of enzymes in sugar metabolism were mostly not affected by elevated CO2 under MN condition, but many enzymes in sugar catabolic pathways were up-regulated by elevated CO2 under LN condition, including beta-glucosidase, invertase, endo-1,3-beta-glucanase, and beta-amylase (Figure 7). Enzymes in the amino acid metabolism were affected differently by elevated CO2 under different nitrate rates. Proteins for enzymes leucine, lysine, and homocysteine synthesis, including 3-isopropylmalate dehydratase large subunit, diaminopimelate

epimerase,

Diaminopimelate

decarboxylase,

and

DNA

(cytosine-5)-methyltransferase were up-regulated by elevated CO2 under MN conditions, but were not affected or down-regulated in response to elevated CO2 under LN conditions (Figure 8). Many proteins involved in protein translation were up-regulated by elevated CO2 under MN conditions, but were either not affected or down-regulated by CO2 under LN conditions, including the components of snoRNP complex (NHP2-like protein 1, mediator of RNA polymerase II transcription subunit 36b, and nucleolar protein 5-2), one ribosomal protein (60S acidic ribosomal protein P0) and two kinds of tRNA ligase. In addition, importin functioning in the transport of proteins into nucleus and importin subunit alpha-1a (COP1) were up-regulated under MN conditions but down-regulated under LN conditions by elevated CO2 (Figure 9). As shown in the Table 1, MCM proteins were related to the DNA replication. Three MCMs were up-regulated by elevated CO2 under MN condition and only two of them were found up-regulated by elevated CO2 under LN condition. Seven molecular chaperones were identified with different abundance between ambient and elevated CO2 under MN conditions, while three of which were up-regulated and the rest were down-regulated by elevated CO2 concentration. Of the seven chaperone proteins, only two DEPs were found under LN conditions, with one up-regulated and the other down-regulated by elevated CO2. 14


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DISCUSSION Leaf growth rate is governed by cell expansion and cell division28. In this study, elevated CO2 enhanced leaf elongation under sufficient N conditions, which was mainly due to increased cell number rather than the elongation of individual cells. The promotive effects of elevated CO2 on leaf growth and cell number were suppressed by low nitrate supply. These results suggested that elevated CO2 and nitrate availability interactively affect leaf growth in tall fescue mainly due to alteration of cell number rather than cell length. Increased cell numbers were regulated by genes and proteins involved in cell cycles and DNA replication. Some cell-cycle genes are the target genes of Multi-copy Suppressor of IRA 1)-like protein (MSI) which is a component of multi-protein complex functioning in chromatin modifications.29,30 In this study, MSI protein was up-regulated by 40% by elevated CO2 when N supply was sufficient and not changed when the N supply was limited. In addition, three MCM (mini-chromosome maintenance) proteins (homologues to OsMCM2, OsMCM3, and OsMCM7) associated with DNA replication were enhanced by elevated CO2 under both nitrate rates, but the degree of up-regulation by elevated CO2 was higher under MN condition than under LN condition. MCM proteins exhibit DNA helicase activity and are required for the initiation and elongation steps of DNA replication.31 In addition, DNA methylation by which the methyl groups are added to DNA and the activity of DNA segments may change without changing the sequence32 may also play roles in regulating plant growth and development.33, 34, 35 Three DNA methyltransferase genes were

found

in

Arabidopsis,

including

MET1,

CMT3

(DNA

(cytosine-5)-methyltransferase), and DRM, while among the three genes, CMT3 is unique to the plant kingdom and involved in maintaining methylation marks through DNA replication.36 The mutants of OsCMT3a gene exhibited developmental abnormalities.37 In this study, a protein homologous to OsCMT3 was up-regulated by elevated CO2. Our results suggested that the enhanced leaf growth by elevated CO2 may be related to changes in the expression of cell-cycle and DNA-replication related 15



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proteins. Plant hormones play crucial roles in regulating cell division and elongation governing leaf growth rate controlling cell numbers. Cytokinin is considered to be the primary hormone controlling cell division in plant growth and development.38 GA and IAA mainly regulate cell elongation, and can also affect cell division.39

40

ABA is

known as a growth inhibitor for leaf elongation.41 Previous studies reported increased content of IAA, GA3, ZR, DHZR and iPA, but decreased ABA content in shoots of Arabidopsis and tobacco plants exposed to elevated CO2.42 43 In this study, the content of iPA, GA and IAA increased and ABA content decreased in tall fescue leaves due to elevated CO2 only when plants were supplied with sufficient nitrate, suggesting that the interactive effects of elevated CO2 and nitrate on leaf growth governed by cell division may involve hormonal regulation. Elevated CO2 led to the down-regulation of a protein (CL4685.Contig3) homologous to OsLEA3 in this study. LEA3 is a member of LEA (late embryogenesis abundant) proteins which is typically up-regulated or induced by ABA and various abiotic stresses44,45 in leaves with restricted growth by ABA.46 In this study, the down-regulation of LEA3 (CL4685.Contig3) by elevated CO2 in tall fescue leaves along with the decline in ABA content suggested that elevated CO2 effects on leaf growth may involve repressing ABA signaling. In addition, ASR proteins have been reported to be regulated by GA47,48 with the protein level of ASR5 in rice significantly increased by GA3.49 In this study, a protein (Unigene29877) homologous to OsASR5 was up-regulated by elevated CO2 in tall fescue leaves, which corresponded with the increased content of endogenous GA content. Our results suggest that leaf growth regulated by CO2 also involve proteins for hormonal signaling or responses. Proteomic analysis of elongating leaves of tall fescue in this study also found that a large proportion of proteins (54 out of 186) differentially accumulated due to elevated CO2 under different nitrate rates were localized in the chloroplast and involved in light harvesting process of photosynthesis. Protein abundance of enzymes in the chlorophyll a synthesis pathway, including CHLH, PORA, and CHLP and most 16


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proteins involved in the light harvesting system, including three PSI reaction center proteins (PsaA, PsaD, and PsaN) and two antenna proteins (CAB1R，Chlorophyll a-b binding protein 1 and LHC1) were up-regulated by elevated CO2 in plants supplied with sufficient nitrate (MN treatment). However, the abundance of most light-harvesting proteins was either unchanged or decreased in plants with low nitrate supply (LN treatment), including five PSII reaction center proteins (PsbA, PsbB, PsbC, PsbD, and PsbH). Photosynthesis of nitrate-limited plants have been shown to be less responsive to elevated CO2 than well-fertilized plants.50 It is interesting to note that the protein abundance of enzymes in sugar metabolism was mostly not affected by elevated CO2 under MN condition. The up-regulation of light-harvesting proteins is corresponded to leaf growth promotion by elevated CO2 in plants with sufficient nitrate supply. In contrast, the promotive effects of elevated CO2 on both leaf growth and light-harvesting proteins were diminished in plants with low nitrate supply. These results suggest that light-harvesting process was more regulated by elevated CO2 rather than sugar metabolism proteins in elongating leaves, which together with leaf growth were correspondingly regulated by the interaction of elevated CO2 and nitrate availability. Amino acids are essential constituents of proteins and are also products of protein catabolism. Enzymes in amino acid metabolism were affected differently by elevated CO2 under different nitrate rates. Proteins for enzymes regulating leucine, lysine, and homocysteine synthesis, including 3-isopropylmalate dehydratase large subunit (LeuC), diaminopimelate epimerase (DAPF), Diaminopimelate decarboxylase (LYSA), and CMT3 were up-regulated by elevated CO2 under MN conditions, but were not affected or down-regulated in response to elevated CO2 under LN conditions (Figure 8). Proteins for enzymes involving the protein catabolic process (such as aspartyl protease, aminopeptidase M1-B, and oryzain alpha chain) were not affected by elevated CO2 in MN condition but up-regulated in LN condition (Table 1). These results indicated that elevated CO2 concentration promoted amino acid synthesis and had no effects on protein catabolism when plants were supplied with adequate nitrate; 17



in contrast, with low nitrate supply, protein catabolism could be activated with elevated CO2, which could enhance breakdown of proteins for providing amino acids to support plant growth under deficient nitrate conditions. In summary, elevated CO2 enhanced leaf growth of tall fescue through the increase of cell number under sufficient N conditions whereas low N supply limited leaf growth responses to elevated CO2. Leaf growth responses to the interaction of elevated CO2 and nitrate availability was associated with changes in the endogenous content or related response proteins for IAA, GA, IPA, or ABA in leaves of tall fescue. The enhanced leaf growth by elevated CO2 supplied with sufficient N was associated with the increased abundance of proteins involved in photosynthesis, amino acid metabolism, DNA replication and translation in elongating leaves of tall fescue.

SUPPORTING INFORMATION: Table S1

The common DEPs between two CO2 levels in the LN and MN conditions

Table S2

The specific DEPs between two CO2 levels in the MN condition

Table S3

The specific DEPs between two CO2 levels in the LN condition

Table S4

The summary of protein quantitation

ACKNOWLEDGEMENTS This work was supported by the program of the program of the Fundamental Research Funds for the Central Universities (KYZ201673), Natural Science Foundation of Jiangsu Province (BK20181320), and National Natural Science Foundation of China (31672480). CONFLICT OF INTEREST STATEMENT The authors have declared no conflict of interest. 18


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Table 1 Selected DEPs responsive to elevated CO2 from tall fescue leaves under different nitrogen supplies. Ratio (eCO2/aCO2) LN

Protein Protein name

MN

accession

ND

1.79

Q2R482

DNA replication licensing factor MCM2 (Minichromosome maintenance protein 2)

1.39

1.73

Q0DHC4


1.43

1.82

Q2QNM1


ND

1.41

Q10G81

Histone-binding protein MSI1 (Multi-copy Suppressor of IRA 1) homolog

0.61

1.22

Q71VM4

Importin subunit alpha-1a (Isa 1a)

0.58

ND

Q9SLX0

Importin subunit alpha-1b (Isa 1b)

1.20

Q10NY2

TPR3 (Topless-related protein 1)

1.44

Q0DWC1

Cell Cycle

Cell Proliferation

Transcription ND Translation Med36b (Mediator of RNA polymerase II transcription subunit 36b), putative 0.56

fibrillarin

25


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

ND

2.05

Q10PA5

NHP2L1 (NHP2-like protein 1)

ND

1.26

Q0DRW2

Nop5-2 (Nucleolar protein 5-2)

ND

1.64

Q10KP0

CP29B (RNA-binding protein CP29B)

ND

1.36

P93422

HisRS (Histidine--tRNA ligase)

ND

1.23

B9FSH5

AlaRS (Alanine--tRNA ligase)

ND

1.24

P41095

RPLP0 (60S acidic ribosomal protein P0)

ND

0.72

P0C477

30S ribosomal protein S18, chloroplastic, rps18

ND

0.58

Q75J18

60S ribosomal protein L13a-2, putative

1.31

0.81

Q10LN7

60S ribosomal protein L18, putative

0.12

ND

C7IZ82

40S ribosomal protein S30 (Fragment)

0.76

ND

P0C482

30S ribosomal protein S2, chloroplastic

0.58

ND

Q0D7V1

Os07g0207400 protein (Fragment)

0.64

ND

Q10NM5

50S ribosomal protein L4

0.35

ND

Q10PV6


0.54

ND

Q6H8H3

Putative plastid ribosomal protein L19

0.46

ND

Q7F4T2


0.77

ND

C7J1A6


26


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0.51

ND

Q75IA5

30S ribosomal protein S13

0.37

ND

P0C491


0.80

ND

Q2R4A1


0.32

ND

Q7XVZ0


0.56

ND

Q0JFH5

Eukaryotic translation initiation factor 3 subunit M (eIF3m)

0.54

ND

Q69UI8

Putative acidic ribosomal protein P1a

0.45

ND

P0C485

30S ribosomal protein S3, chloroplastic

0.69

ND

Q0JEI2


0.46

ND

Q60E59

Ribosomal protein

0.24

ND

C7IYE9


ND

1.90

P12330

CAB1R (Chlorophyll a-b binding protein 1)

0.64

1.96

Q69P91

LHCI (Chlorophyll a-b binding protein)

0.63

ND

Q84NW1

ATPC1 (ATP synthase gamma chain 1)

ND

1.37

Q6Z2T6

CHLP (Geranylgeranyl diphosphate reductase)

ND

2.25

Q7XKF3

PORA (Protochlorophyllide reductase A)

ND

1.22

Q10M50

CHLH (Magnesium-chelatase subunit H)

Photosynthesis

27



0.83

1.36

P0C355

psaA (Photosystem I P700 chlorophyll a apoprotein A1)

0.80

1.26

Q84PB4

psaD (Photosystem I 20 kDa subunit)

0.68

ND

Q0DG05

psaH (Photosystem I reaction center subunit VI)

0.71

1.75

Q2QWN3

psaN (Photosystem I reaction centre subunit N)

0.74

ND

P0C434

psbA (Photosystem II protein D1)

0.68

ND

P0C364

psbB (Photosystem II CP47 reaction center protein)

0.70

ND

P0C367

psbC (Photosystem II CP43 reaction center protein)

0.61

ND

P0C437

psbD (Photosystem II D2 protein)

0.69

ND

P0C422

psbH (Photosystem II reaction center protein H)

ND

1.27

Q5ZDI7

psbP domain-containing protein 5

Amino Acid Metabolism ND

1.61

Q9ASP4

Dihydrolipoyl dehydrogenase

ND

0.76

Q0JEK1

DHDPS (4-hydroxy-tetrahydrodipicolinate synthase 1)

ND

0.59

Q2QMG2

MCCA (Methylcrotonoyl-CoA carboxylase subunit alpha)

ND

0.64

Q7XPR2

Aminomethyltransferase

ND

0.45

Q10CE7

GSTU1 (glutathione S-transferase)

ND

1.52

Q6Z702

LeuC (3-isopropylmalate dehydratase large subunit)

28


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ND

1.51

Q6ZG77

LYSA (diaminopimelate decarboxylase)

ND

1.90

C0SQ89

CMT3 (DNA (cytosine-5)-methyltransferase)

0.50

1.43

Q2QNF7

DAPF (Diaminopimelate epimerase)

0.81

0.65

Q10D68

Serine hydroxymethyltransferase

0.78

0.58

Q69RJ0

GLU (Ferredoxin-dependent glutamate synthase)

3.81

0.82

Q0ISV7

SAHase (Adenosylhomocysteinase)

ND

1.55

P37833

AST (Aspartate aminotransferase)

0.61

0.44

O64422

FBP (Fructose-1,6-bisphosphatase)

1.53

ND

Q75I93

BGLU (Beta-glucosidase 7)

1.61

0.37

Q8S9Q6

GNS (endo-1,3-beta-glucanase)

1.61

ND

Q10RZ1

BAMY (Beta-amylase 2)

0.56

0.20

Q0JKY8

Carbonic anhydrase

ND

1.54

Q2R8Z5

ADH1 (Alcohol dehydrogenase 1)

ND

0.56

Q5JLP6

S-formylglutathione hydrolase

1.71

ND

Q0JBF1

Inv (Invertase，beta-fructofuranosidase)

0.52

ND

Q69V57

Fructose-bisphosphate aldolase

Sugar Metabolism

29



0.48

ND

Q40677

0.71

ND

Q0DCI1

Fructose-bisphosphate aldolase PFP-ALPHA (Pyrophosphate-fructose 6-phosphate 1-phosphotransferase subunit alpha)

Protein Catabolic Process 1.50

ND

P25776

Oryzain alpha chain

1.64

ND

Q6F4N5

Aspartyl protease 25

1.68

ND

Q0JG83

Os01g0937000 protein

2.04

ND

Q10M95

Eukaryotic aspartyl protease family protein, expressed

1.60

ND

Q5Z4E5

Putative 41 kD chloroplast nucleoid DNA binding protein

1.54

ND

Q0J5V5

Aminopeptidase M1-B (Alpha-aminoacylpeptide hydrolase)

1.47

0.76

Q10RJ2

Hsp20 (20 kDa heat shock protein)

0.47

1.26

Q2QV45

Hsp70 (70 kDa heat shock protein)

ND

0.66

Q07078

HSP81-3 (Heat shock protein 81-3)

ND

0.64

Q0D9G9

endoplasmin homolog

ND

0.80

Q2QU06

Chaperonin

ND

1.36

Q6ASR1

Chaperonin

Protein Folding

30


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ND

1.31

Q5Z907

Chaperonin

1.25

1.99

Q5U1Q4

prx39 (Peroxidase)

1.71

ND

Q0D3N0

Peroxidase

1.29

ND

Q6AVZ3

Peroxidase

1.25

ND

Q7XSV1

Peroxidase

1.77

ND

Q652L6

MDAR3 (Monodehydroascorbate reductase 3)

ND

2.49

Q9FE01

APX2 (Ascorbate peroxidase)

0.66

1.22

Q6ER94

BAS1 (2-Cys peroxiredoxin)

1.49

2.02

Q10F62

thiC (Thiamine biosynthesis protein)

1.26

3.26

Q7XXS4

THI1 (Thiamine thiazole synthase)

1.49

0.35

Q10MP7

Pathogenesis-related protein 1

0.77

0.66

Q7EYM8

Putative oxidoreductase

ND

0.75

P0C5A4

LEA3 (Late embryogenesis abundant protein, group 3)

ND

1.32

Q53JF7

ABA/WDS induced protein

Response to Stress

Response to ABA

Isoprenoid biosynthesis

31



ND

1.28

Q6K8J4

ISPG (4-hydroxy-3-methylbut-2-enyl diphosphate synthase)

ND

1.69

Q6AVG6

ISPH (4-hydroxy-3-methylbut-2-enyl diphosphate reductase)

ND: not detected.

32


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Figure legends Figure 1. Shoot growth characteristics of tall fescue as affected by atmospheric CO2 concentrations and NO3- supplies. A, the shoot of tall fescue under 40 d treatment; B, leaf length; C, Shoot biomass. aCO2, ambient CO2; eCO2, elevated CO2; LN, 0.25 mM NO3-; MN, 4 mM NO3-; HN, 32 mM NO3-. Figure 2. Effects of elevated CO2 on tall fescue cell lengths (A) and numbers (B) of mature leaves under different NO3- supplies. aCO2, ambient CO2; eCO2, elevated CO2; LN, 0.25 mM NO3-; MN, 4 mM NO3-; HN, 32 mM NO3-. Figure 3. Content of endogenous phytohormones from tall fescue leaves as affected by atmospheric CO2 concentrations and NO3- supplies. aCO2, ambient CO2; eCO2, elevated CO2; LN, 0.25 mM NO3-; MN, 4 mM NO3-; HN, 32 mM NO3-. Figure 4. Venn diagrams showing numbers of differentially-expressed proteins (DEPs) identified from tall fescue leaves exposed to different atmospheric CO2 concentrations. Proteins showing up-regulation (A), and down-regulation (B). aCO2, ambient CO2; eCO2, elevated CO2; LN, 0.25 mM NO3-; MN, 4 mM NO3-. Figure 5. Numbers of differentially expressed proteins (DEPs) identified from tall fescue leaves under different CO2 levels in sufficient nitrogen supply (MN, 4 mM NO3-) and limited nitrogen supply (LN, 0.25 mM NO3-). The proteins were classified based on their predicted participated biological process (A), molecular functions (B), and cellular components (C). Figure 6. Schematic diagram of photosynthetic pathways in tall fescue showing representative differentially displayed proteins. The metabolic pathways were constructed based on the proteomic data, and the broken arrows indicate that there are multi-steps between the two compounds. The up regulated and down regulated DEPs were marked with red and blue circles (LN) or square (MN), respectively. CHLH, Magnesium-chelatase subunit H, PORA, Protochlorophyllide reductase A, CHLP, Geranylgeranyl diphosphate reductase, CABIR, Chlorophyll a-b binding protein 1, PsbA, Photosystem II protein D1, psbB, Photosystem II CP47 reaction center protein, 33



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psbC, Photosystem II CP43 reaction center protein, psbD, Photosystem II D2 protein, psbH, Photosystem II reaction center protein H, psbP, psbP domain-containing protein 5, psaA, Photosystem I P700 chlorophyll a apoprotein A1, psaD, Photosystem I 20 kDa subunit, psaH, Photosystem I reaction center subunit VI, psaN, Photosystem I reaction centre subunit N, LHCI, Chlorophyll a-b binding protein, ATPC1, ATP synthase gamma chain 1, PSI, photosynthesis I, PSII, photosynthesis II. Figure 7. Schematic diagram of sucrose and starch metabolism in tall fescue showing representative differentially displayed proteins. The metabolic pathways were constructed based on the proteomic data, and the broken arrows indicate that there are multi-steps between the two compounds. The up regulated and down regulated DEPs were marked with red and blue circles LN or square MN, respectively. BGLU, Beta-glucosidase 7, FBP, Fructose-1,6-bisphosphatase, Inv, Invertase,

beta-fructofuranosidase,

GNS,

endo-1,3-beta-glucanase,

BAMY,

Beta-amylase 2. Figure 8. Schematic diagram of amino acid metabolism in tall fescue showing representative differentially displayed proteins. The metabolic pathways were constructed based on the proteomic data, and the broken arrows indicate that there are multisteps between the two compounds. The up regulated and down regulated DEPs were marked with red and blue circles LN or square MN, respectively. LeuC,3-isopropylmalate

dehydratase

large

subunit,

LYSA,

diaminopimelate

decarboxylase, CMT3,DNA (cytosine-5)-methyltransferase, DAPF, Diaminopimelate epimerase, GLU, Ferredoxin-dependent glutamate synthase, AST, Aspartate aminotransferase,

SAHase,

Adenosylhomocysteinase,

SAM,

S-Adenosyl-L-methinonine. Figure 9. Schematic diagram of translation process in tall fescue showing representative differentially displayed proteins. The metabolic pathways were constructed based on the proteomic data, and the broken arrows indicate that there are multisteps between the two compounds. The up regulated and down regulated DEPs were marked with red and blue circles LN or square MN, respectively. Isa 1a,Importin 34


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subunit alpha-1a, Isa 1b, Importin subunit alpha-1b,

Med36b, Mediator of RNA

polymerase II transcription subunit 36b ( putative fibrillarin), NHP2L1, NHP2-like protein 1, Nop5-2, Nucleolar protein 5-2, CP29B, RNA-binding protein CP29B, HisRS, Histidine--tRNA ligase, AlaRS, Alanine--tRNA ligase, RPLP0, 60S acidic ribosomal protein P0, NPC, nuclear pore complex.

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For TOC Only

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Fig. 1 80x77mm (300 x 300 DPI)



Fig. 2 254x119mm (300 x 300 DPI)


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Fig. 3 319x219mm (300 x 300 DPI)



Fig. 4 180x80mm (300 x 300 DPI)


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Fig. 5 338x190mm (96 x 96 DPI)



Fig. 6 279x119mm (300 x 300 DPI)


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Fig. 7 163x135mm (300 x 300 DPI)



Fig. 8 149x119mm (300 x 300 DPI)


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Fig. 9 160x116mm (300 x 300 DPI)


Proteomic profiling for metabolic pathways involved in interactive

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