iTRAQ protein profile analysis of young and old leaves of cotton (Gossypium hirsutum L.) differing in response to alkali stress

Plant Physiology and Biochemistry 141 (2019) 370–379

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Research article

iTRAQ protein profile analysis of young and old leaves of cotton (Gossypium hirsutum L.) differing in response to alkali stress

T

Yongjun Hub, Long Zhaoc, Ji Zhoud, Xiuli Zhonga, Fengxue Gua, Qi Liua, Haoru Lia, Rui Guoa,∗ a Key Laboratory of Dryland Agriculture, Institute of Environment and Sustainable Development in Agriculture, Chinese Academy of Agricultural Sciences, Beijing, 100081, China b School of Life Sciences, ChangChun Normal University, Changchun, 130024, China c Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China d Land Consolidation and Rehabilitation Centre, Ministry of Natural Resources of the People's Republic of China, Beijing, 100000, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: Gossypium hirsutum L Young leaves Old leaves iTRAQ Proteomics Alkali stress

Proteins will provide a new perspective and deeper understanding for the research of crop alkali tolerance. The aims of this study were to determine and identify the differentially abundant proteins and adaptive mechanisms to alkali tolerance between young and old leaves of cotton. The 4704 proteins were identified, in which 1490 were significantly changed in young leaves and 563 were changed in old leaves in response to alkali stress. The differentially abundant proteins were classified into 10 functional categories in the young leaves, and only 3 functional categories were involved in the old leaves. In the photoreaction system, the accumulations of differential proteins, especially Psb proteins, were higher in young leaves than in old leaves. Compared with old leaves, the carbon metabolism was enhanced significantly through an increased chlorophyll content and increased expression of key proteins for carbon metabolism in young leaves. Furthermore, alkali stress revealed more complex effects on the nitrogen metabolism in young leaves than that in old leaves. Our results demonstrated that during adaptation of cotton to alkali stress, young and old leaves have distinct mechanisms of molecular metabolism regulation. The metabolic flexibility was more remarkable in young leaves than in old leaves; therefore, the alkali tolerance of young leaves is more efficient. These data will increase our understanding of alkali-tolerant mechanisms in higher plants.

1. Introduction

salt stress have been analyzed including model plants Arabidopsis; grain crops, such as rice; wheat; cash crops, such as tomatoes; tobacco; and halophytes, such as salt mustard (Dani et al., 2005; Jiang et al., 2007; Wang et al., 2008; Chen et al., 2009a; Pang et al., 2010; Wen et al., 2010). Studies have found that, unlike in halophytes, the salt stress-responsive proteins of most glycophytes are mainly involved in photosynthesis, carbon and energy metabolism, stress defense, basal metabolism, and signal transduction. Moreover, the salt stress response proteome changes of 13 glycophytes, including model plant Arabidopsis and rice, have been analyzed (Millar and Heazlewood, 2003). In 81 salt stress-responsive proteins of Arabidopsis roots, about 52% of them are involved in carbon and energy metabolism (e.g. tricarboxylic acid cycle, glycolysis and electron transfer processes), stress defense, and protein metabolism; whereas in photosynthetic tissues, the expression patterns of photosynthesis and basal metabolism-related proteins change significantly (Jiang et al., 2007; Pang et al., 2010). Meanwhile, studies have found that abscisic acid (ABA) and gibberellin

Salinity affects over 6% of land areas around the world, which seriously affects the growth and development of plants, limits the regional distribution of plants, and restricts the quality and yield of plants (Flowers et al., 2010; Wang et al., 2011). After plants ingest high concentrations of salt ions, osmotic stress, ionic stress and oxidative stress occur, which then affects the metabolism of substances and energy in plants and inhibits the growth of plants or even lead to death (Munns, 2002). Recent advances in techniques for high-throughput proteomics have enabled the analyses of functions of plant proteins and stress treatmentrelated changes to protein expression levels (Schwacke et al., 2009; Jiang et al., 2015). Therefore, proteomic analyses could provide more accurate and comprehensive insights into transcriptional, translational, and post-translational levels (Rutschow et al., 2008; Kondrák et al., 2011). In recent years, the proteome changes of various plants under

∗

Corresponding author. E-mail address: [email protected] (R. Guo).

https://doi.org/10.1016/j.plaphy.2019.06.019 Received 9 May 2019; Received in revised form 13 June 2019; Accepted 13 June 2019 Available online 14 June 2019 0981-9428/ © 2019 Elsevier Masson SAS. All rights reserved.

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during the nighttime. After 4 weeks, the six pots were randomly divided into two sets. The first set of pots was watered with Hoagland's nutrient solution daily as before, while the other set was watered with 80 mM alkali stress solution containing 72 mM NaHCO3 and 8 mM Na2CO3.The young leaves used were the second leaves from the top, and the old leaves used were the bottom leaves. The seedlings were harvested and stored at −80 °C after 6 d of treatment. One seedling per pot served as a biological replicate and each treatment had three biological replicates.

(GA) can change the expression patterns of 50 proteins in rice seedlings, such as molecular chaperones, salt-stress induced protein (SALT protein), ribulose-1,5-diphosphate carboxylase/oxygenase (RuBisCO), and phosphate glucose translocation enzyme, and can improve the tolerance of plants to salt stress by regulating photosynthesis, energy metabolism, protein metabolism, signal transduction, and stress defense (Li et al., 2010; Wen et al., 2010). In addition, salt-responsive proteomics analyses of food crops, such as wheat, soybean, and cash crops, such as potato, peanut, tobacco, and grape, have shown that most salt stressresponse proteins of glycophytes are mainly involved in photosynthesis, carbon and energy metabolism, stress defense, basal metabolism, and signal transduction (Dani et al., 2005; Jain et al., 2006; Aghaei et al., 2008, 2009,; Jellouli et al., 2008; Wang et al., 2008; Peng et al., 2009). It is well known that salinity stress has different effects on different tissues though effects on the growth, compatible solutes accumulation and ion metabolism, especially in young and old tissues (Munns and Tester, 2008; Hajlaoui et al., 2010; Wang et al., 2012a; Guo et al., 2016). Saline-alkaline soil has a wide range of concentrations and types of salts and contains predominant ions, including Na+, Ca2+, K+, Cl−, SO42−, and CO32− (Shi and Wang, 2005). Salinity stress has been classified into salt and alkali stresses based on the particular mix of salts present (Shi and Wang, 2005; Yang et al., 2007). Generally, salt stress was simulated by mixing NaCl and Na2SO4 and alkali stress was simulated by mixing NaHCO3 and Na2CO3. Salt stress includes osmotic stress and ion injury, whereas alkali stress includes both, but features a high pH value (Shi and Wang, 2005). The high-pH cause metal ions and phosphorus to precipitate, and the loss of the normal physiological functions of the tissues, even the destruction of cell structure (Shi and Wang, 2005; Yang et al., 2007; Guo et al., 2017). Therefore, alkali stress has stronger injurious effects on plants than salt stress. Although attention has been given to studying alkali stress on different tissues, available data on the comparative study of old and young tissues are lacking. Young and old tissues may play different roles in alkali tolerance, the understanding of comparative effects of alkali stress on young and old tissues may be important for alkali tolerance research. Cotton (Gossypium hirsutum L.) is an important economic crop; it is the earliest commercialized transgenic crop and is worth approximately $56 billion in the world (Brookes and Barfoot, 2013). Cotton is frequently used as a model crop to investigate resistance to different stresses (i.e., salinity, alkalinity, drought, and cold), gene flow, and genetic variation among clones and populations, as well as coordination of growth and biomass allocation strategy (James, 2009; Zhang et al., 2015; Wang et al., 2012b; Guo et al., 2016). Leaves are important in photosynthesis, the functional characteristics of leaves varies with different age, and affecting the exchange of substances and energy between plants and the surrounding environment (Diepenbrock, 2010). In our study, we compared alkali stress-induced quantitative and qualitative changes in proteomes in young (the second leaf at up) and old (the first leaf at bottom) leaves of cotton seedlings using isobaric tags for relative and absolute quantitation (iTRAQ) technique in order to identify differentially abundant proteins involved in alkali tolerance and to reveal the distinct molecular mechanisms underlying young and old leaves’ responses to alkali stress.

2.2. Chlorophyll content 500 mg fresh leaves materials were randomly sampled to determine chlorophyll content in acetone extracts spectrophotometrically as described by Zhu (1993). Each test was repeated thrice. 2.3. Protein preparation including protein extraction, trypsin digestion, peptide isobaric labeling The cotton leaves were snap-frozen in liquid nitrogen and ground into a powder. For protein extraction, the samples were added with 1:50 (W/V) Lysis Buffer (8 M urea, 2 mM EDTA, 10 mM DTT and 1% Protease Inhibitor Cocktail) and homogenized thoroughly with a tissue grinder. Samples were sonicated and centrifuged at 15000 g at 4 °C for 10 min, then the protein in supernatant was determined using a Modified Bradford Protein Assay Kit according to the manufacturer's instructions. For digestion, 100 μg protein of each sample was first reduced with 10 mM DTT at 37 °C for 60 min and then alkylated with 55 mM iodoacetamide (IAM) at room temperature for 30 min in darkness. The protein pool of each sample was digested with Sequencing Grade Modified Trypsin with the ratio of protein: trypsin = 50: 1 mass ratio at 37 °C overnight and 100:1 for a second digestion for 4 h. After trypsin digestion, peptide was desalted by Strata X SPE column and vacuum-dried. Peptide was reconstituted in 20 mm3 500 mM TEAB and processed according to the manufacturer's protocol for 8-plex iTRAQ kit. 2.4. LC-MS/MS identification The dried and labeled peptide was reconstituted with high-performance liquid chromatography (HPLC) solution (2 %ACN, pH 10) and then fractionated into fractions by high pH reverse-phase HPLC using Waters Bridge Peptide BEH C18 (130 Å, 3.5 μm, 4.6*250 mm).The peptides were combined into 10 fractions. Then the experiment was performed by NanoLC 1000 LC-MS/MS using a Proxeon EASY-nLC 1000 coupled to Thermo Fisher Q Exactive. Trypsin digestion fractions were loaded onto delivered to a reversed-phase pre-column (Acclaim PepMap®100 C18, 3 μm, 100 Å, 75 μm × 2 cm) and a reversed-phase analytical column (Acclaim PepMap® RSLC C18, 2 μm, 100 Å, 50 μm × 15 cm). The gradient was comprised of an increase from 15% to 35% solvent B (0.1% FA in 98% ACN) over 45 min, 35%–98% solvent B during 5 min and keep in 98% in 5 min at a constant flow rate of 300 nl/min on an EASY-nLC 1000 system. The eluent was sprayed via NSI source at the 2.0 kV electrospray voltage and then analyzed by tandem mass spectrometry (MS/MS) in Q Exactive. The mass spectrometer was operated in data-dependent mode, automatically switching between MS and MS/MS. Full-scan MS spectra (from m/z 350 to 1800) were acquired in the Orbitrap with a resolution of 70,000. Ion fragments were detected in the Orbitrap at a resolution of 17,500, the 20 most intense precursors were selected for subsequent decision treebased ion trap HCD fragmentation at the collision energy of 30% in the MS survey scan with 45.0 s dynamic exclusion.

2. Materials and methods 2.1. Plant materials All seeds of cotton (Gossypium hirsutum L.) Yiluzao-7 used in this study were received from the National Cotton Improvement Center, Cotton Research Institute, Chinese Academy of Agricultural Sciences, Beijing, China. This study was carried out from May to July in 2018. The cotton seeds were sown in six pots, with each pot containing five seedlings. All seeds were grown outdoor and protected from rain. They were sufficiently watered with Hoagland's nutrient solution daily. The temperature were 23°C–27 °C during the daytime and 19 °C–22 °C

2.5. Protein function annotation and data analyses Protein identification and quantification in the isobaric tag for relative and absolute quantitation (i TRAQ experiment) was performed 371

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Although 154 (8.7%) up and 43 (2.4%) down-regulated proteins were conserved between young and old leaves, 86 (4.8%) DAPs had opposite expression patterns in leaf samples from the two developmental stages (Fig. 1 C). Indeed, 48 (2.7%) proteins that were up-regulated in young leaves were down regulated in old leaves, and 38 (2.1%) proteins showed the opposite expression trend (Fig. 1 C). In addition, the number of DAPs in young leaves was much greater than in old leaves (Fig. 1 C). The number of up-regulated proteins in young leaves (735) was twice that in old leaves (371), and, for down-regulated proteins, the ratio (755–192) was four times greater (Fig. 1 C). DAPs were screened from primary protein data using the t-test (P < 0.05) and FC values (fold > 1.5).

using MaxQuant (1.3.0.5 version). For protein identification, the cotton (G. hirsutum) protein database of NCBI (https://www.ncbi.nlm.nih.gov/ , downloaded at Aug 2015) which have 78527 proteins totally was used with the criterion of a false discovery rate (FDR) < 0.01. The parameters of library searching were as following: Carboxymethylation on cysteine residues was set as a fixed modification; variable modifications include iTRAQ 8plex labeling on the N-terminus and lysine, oxidative modification on methionine. As a result, 4704 proteins, with more than two peptide sequences, were identified, including 4664 proteins, that have information in all eight iTRAQ quantitative channels. The uniprot database (http://www.uniprot.org/) was also used for annotations. For sample comparisons, proteins with p values < 0.05, fold change > 1.5 were considered to be significant. For the gene ontology (GO) annotation, Blast2Go software (https://www.blast2go.com/) using the BLASTp search algorithm was used, with a criterion of an E-value less than 1e−5, to search the NR database. The Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation was performed using the KAAS database (https://www.genome.jp/tools/kaas/) and the bi-directional best hit method to assign orthologs. For GO and KEGG enrichment analyses and the dotplot visualization, clusterprofiler (Yu et al., 2012), an R bioconductor package, was used, with a criterion of an adjusted P value < 0.05. R (3.5.1 version) software was used for the statistical analyses. The KEGG pathway figures were generated based on KEGG database (https://www.genome.jp).

3.3. GO and KEGG enrichment analysis The roles of alkali-stress-induced DAPs in cotton were investigated using GO and KEGG pathway enrichment analyses. For both of analyses, the “BH” method was used to adjust P values, and 0.05 was used as the threshold. Intriguingly, for the GO and KEGG analyses, the young and old leaves performed almost completely differently, with no overlap (Figs. 2 and 3; Table 2). For the GO analyses, the DAPs in old leaves were enriched in “ATP hydrolysis coupled proton transport”, whereas those in the young leaves were significantly enriched in “oxidoreductase activity”, “carbohydrate metabolic process” and, “pyridoxal phosphate binding”, with DAPs related to “integral component of membrane” and “metal ion binding” also contributing large percentages (Fig. 2). For down-regulated DAPs, “hydrogen peroxide catabolic process”, “extracellular region”, “peroxidase activity”, “chloroplast” and “response to oxidative stress” were most abundant in old leaves, while “structural constituent of ribosome”, “translation”, “GTP binding”, as well as “GTPase activity” were predominantly down-regulated in young leaves (Fig. 2). For the KEGG enrichment analyses, DAPs were enriched in 19 pathways in young leaves, with 12 and 7 pathways being enriched with respect to up-regulated and down-regulated DAPs, respectively (Table 2). All the up-regulated DAPs were enriched in metabolic pathways, especially in the carbohydrate and energy metabolism pathways, whereas cellular processes and genetic information processing pathways were the main pathways associated with down-regulated DAPs (Table 2). A total of four metabolic pathways were significantly enriched in old leaves, and the most abundant up-regulated DAPs were enriched in “oxidative phosphorylation (ko00190)”. We did not discover any down-regulated DAPs enriched in the metabolic pathways (Table 2). By comparing the KEGG enrichment result, we determined that young and old leaves had diverse responses to alkali stress, including opposite responses (Fig. 3). Some proteins in young leaves were upregulated in several specific pathways, whereas these proteins were down-regulated in the same pathways in old leaves, and vice versa (Fig. 3). In detail, proteins involved in fructose and mannose metabolism (ko00051), carbon fixation in photosynthetic organisms (ko00710), methane metabolism (ko00680), and carbon metabolism

3. Results 3.1. Chlorophyll content Alkali stress revealed different effects on the chlorophyll contents of young and old leaves (Table 1, P < 0.05). Compared with the control, the alkali stress induced an increase in the chlorophyll content in young leaves, while it decreased the contents of old leaves (Table 1, P < 0.05). The alkali stress also significantly altered the chlorophyll a/ b ratio in young leaves (Table 1, P < 0.05). 3.2. Primary data analysis and identification of differentially accumulated proteins (DAPs) Using iTRAQ, 4704 proteins with more than two peptide sequences were obtained. These proteins are believed to be reliable and were used for subsequent analyses. Compared with control samples, 1490 and 563 proteins showed significant changes in young and old leaves, respectively, under alkali stress (Fig. 1). The expression patterns of alkalinestress-induced DAPs between young and old samples were similar in the analogous protein-transcriptional levels and FC distributions between the two developmental stages (Fig. 1A and B). The protein accumulation level and FC centralization were between 1500 and 25,000 and between 1.5 and 2, respectively (Fig. 1A and B). However, for individual proteins, the responses to alkali stress were distinct at different developmental stages. Among the DAPs, more than 80% were unique, having no overlap with those of other differential accumulation models.

Table 1 Effects of alkali stress on the contents of chlorophyll a, chlorophyll b, chlorophyll a/b, carotenoid, and the total pigments in young and old leaves of cotton seedlings. The values are the means ± SE of three biological replicates. Significant difference between control and alkali stress in cotton leaves was determined by using t-test and marked as “*” (P < 0.05). Leaves/Treatment

Pigments (mg·g

−1

Young

FW)

Chlorophyll a Chlorophyll b Chlorophyll a/b Carotenoids The total pigments

Old

Control

Alkali stress

Control

Alkali stress

2.67 1.30 2.05 0.65 4.62

3.13 3.39 0.92 1.98 8.50

9.53 ± 0.89* 4.05 ± 0.23* 2.35 ± 0.18 2.04 ± 0.14 15.61 ± 1.58*

6.27 ± 0.67 2.64 ± 0.17 2.37 ± 0.12 1.85 ± 0.10 10.76 ± 0.95

± 0.61 ± 0.04 ± 0.23* ± 0.08 ± 0.83

372

± 0.18* ± 0.19* ± 0.09 ± 0.07* ± 0.63*

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Fig. 1. Distribution of alkaline-stress-induced DAPs. Expression pattern of alkaline-stress-induced DAPs in young leaves samples (A) and old leaves samples (B) of cotton seedlings. Violin plots at the top left corner represent alkaline-stress-induced log2FC of two kinds of samples. ASY, alkaline-stressed young leaves samples; CKY, control young leaves samples; ASO, alkaline-stressed old leaves samples; CKO, control old leaves samples. (C) Overlapping of alkaline-stress-induced DAPs between young and old leaves samples. Y_UP, up-regulated proteins in young leaves samples; Y_DOWN, down-regulated proteins in young leaves samples; O_UP, upregulated proteins in old leaves samples; O_DOWN, down-regulated proteins in old leaves samples. (Venny (Oliveros, 2007–2015) was used for (C)).

Fig. 2. Classification of identified proteins. Gene Ontology (GO) enrichment patterns of alkaline-stress-induced DAPs of young leaves samples and old leaves samples of cotton seedlings. Up-regulated DAPs were in the left, and down-regulated DAPs were in the right.

3.4. Analysis of the identified proteins related to alkali resistance

(ko01200) were up-regulated in young leaves, whereas they were down-regulated in old leaves (Fig. 3A). An opposite protein accumulation pattern was detected in the phagosome pathway (Fig. 3A). In addition, we compared the mean log2FC (stress/control) in 20 pathways (Fig. 3B) and observed that transport and catabolism by means of phagosomes (ko04145) and lysosomes (ko04142), respectively, may be the main alkali stress-response strategies for old leaves. Young leaves did not show greater protein accumulation levels in catabolism (ko04210), but they did in carbohydrate (ko00051, ko00040, ko01200, and ko00630), amino acid (ko00250), and energy metabolism (ko00710 and ko00680) (Fig. 3B). We calculated the accumulation patterns of 19 proteins involved in the carbon metabolism of young and old leaves samples and found that these proteins had considerably, if not completely, contrasting expression patterns between the two developmental stages (Fig. 3C). Indeed, 15 of 19 proteins were up-regulated in young leaves but were down-regulated in old leaves (Fig. 3C), which may contribute to the distinct phenotypes of old and young leaves.

Several protein families that are pivotal in plant alkali-stress resistance were annotated, and their expression levels were calculated. A total of 40 proteins related to the classical plant alkali tolerance pathways were extracted, and 39 of them were defined as DAPs. Thus, they may play a significant role in the cotton alkali-stress strategy (Fig. 4). We divided these proteins into six categories: calcium-related (4 proteins), ion-balance (14 proteins), betaine-related (2 proteins), photosystem (7 proteins), antioxidative-system (5 proteins), and heat shock proteins (8 proteins) (Fig. 4). As expected, 20 of 40 proteins had divergent accumulation patterns in response to alkali stress between young and old leaves. A total of 10 (CALR_1, CALR_2, CANX, ATPeF1G, ATPF1G, psbO, psbQ_2, PYR/PYL, HSP90B_2, and HSPA4) proteins were up-regulated by alkali stress in young leaves but were downregulated or unchanged in old leaves (Fig. 4). By contrast, six proteins (ATPeV0A, PMA1_1, PMA1_3, PMA1_4, betB_2, and psbP_1) were upregulated in old leaves but were down-regulated or unchanged in young 373

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Fig. 3. KEGG enrichment of alkaline-stress-induced DAPs in two kinds of samples. (A) Box plot of alkaline-stress-induced log2FC of proteins accumulation in 20 pathways. (B) Spider chart represents relative accumulation pattern of alkaline-stress-induced DAPs in two kinds of samples. (C) Alkaline-stress-induced differentially accumulated pattern of proteins in carbon metabolism pathway (ko01200).

to stress-resistance strategies in cotton. This finding was illustrated by the number and functional divergence of the DAPs between young and old leaves. As shown in the iTRAQ data, we identified 4704 proteins in young and old leaves that possessed more than two unique peptides. This result may not represent the entire landscape of protein patterns in young and old leaves under stress or control condition owing to the technical limitations of iTRAQ. However, many footprints regarding the responses of cotton to alkali stress. Furthermore, out of the 4704 proteins, we identified 1490 and 563 DAPs in young and old leaves, respectively (File 1). The number of DAPs in young leaves was 3 times higher than in old leaves. This result indicated that, in response to alkali stress, the metabolic flexibility was greater in young leaves than in old leaves. Although 197 DAPs shared the same responses in young and old leaves, 86 DAPs showed opposite trends (Fig. 1C). For either GO and KEGG enrichment analyses, no terms or pathways were significantly enriched in young and old leaves (Figs. 2 and 3; Table 2). The young leaves were more sensitive to alkali stress than old leaves; however, the stress may not threaten essential life activities, such as growth and development, and it may even enhance chlorophyll biosynthesis and

leaves (Fig. 4). ATPeF1B, katE, and HSP90B_1 were down-regulated in young leaves but were unchanged in old leaves, whereas HSP5 had the opposite expression trend (Fig. 4). In general, most proteins in these pathways were up-regulated in cotton, in both young (25, 62.50%) and old (21, 52.50%) leaves (Table 3). In both young and old leaves, ionbalance, betaine-related, and antioxidative-system proteins were upregulated (≥50%) (Table 3). However, compared with old leaves, young leaves had more calcium-related and heat shock proteins, which may play roles as sensors and in the reestablishment of cellular homeostasis, respectively.

4. Discussion 4.1. Divergent responses of young and old leaves to alkali stress Stress may have distinct effects on different organs or tissues in plant; however, the data regarding the effects of different developmental stage are limited (Matsui et al., 2001; Ben-Gal et al., 2009). In this study, the results suggested that young and old leaves may have divergent responses to alkali stress and may make distinct contributions 374

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Table 2 Significantly enriched pathways for DAPs between two treatment in young and old cotton leaves. Tissue

DAPs

Pathway and Description

Young leaves

Up-regulated

Carbohydrate metabolism

Amino acid metabolism

Energy metabolism

Old leaves

Down-regulated

Lipid metabolism Transport and catabolism

Up-regulated

Translation Cellular community Signal transduction Cell growth and death Folding, sorting and degradation Transport and catabolism

Down-regulated

Energy metabolism Signal transduction None

Glycolysis/Gluconeogenesis Pentose phosphate pathway Starch and sucrose metabolism Fructose and mannose metabolism Pentose and glucuronate interconversions Glyoxylate and dicarboxylate metabolism Arginine and proline metabolism Alanine, aspartate and glutamate metabolism beta-Alanine metabolism Carbon fixation in photosynthetic organisms Methane metabolism Linoleic acid metabolism Phagosome Endocytosis Ribosome Gap junction AMPK signaling pathway Apoptosis RNA degradation Phagosome Lysosome Oxidative phosphorylation mTOR signaling pathway None

Pathway ID

Counts

P. adjust

ko00010 ko00030 ko00500 ko00051 ko00040 ko00630 ko00330 ko00250 ko00410 ko00710 ko00680 ko00591 ko04145 ko04144 ko03010 ko04540 ko04152 ko04210 ko03018 ko04145 ko04142 ko00190 ko04150 None

10 8 7 5 3 8 4 4 4 10 6 3 7 6 12 6 6 5 3 9 3 10 4 None

0.00595 0.00595 0.00595 0.00711 0.04095 0.02442 0.00595 0.03849 0.00595 0.00595 0.04095 0.00595 0.01296 0.01749 0.00291 0.00002 0.00025 0.00005 0.04864 0.00042 0.00774 0.00061 0.03863 None

activity in young leaves, but reduced the expression of proteins that respond to oxidative stress in old leaves (Fig. 2). The antioxidant system in young leaves under alkali stress is similar to that in salt-stressed cotton (Desingh and Kanagaraj, 2007; Wang et al., 2016). However, alkali stress severely inhibits the growth of old leaves in cotton, as indicated by the down-regulated PS proteins (Figs. 2 and 3), which lay the foundation for the biological processes required during the plant's life cycles, and the up-regulated proteins related to phagosomes and lysosomes, which may trigger apoptosis (Fig. 3; Table 2). Thus, protein accumulation in young and old leaves are distinct and may contribute to the divergence in their physiological and biochemical responses, implying that the diverging accumulations might be an adaptive strategy of plants to alkali stress (Table 4). 4.2. Carbon and nitrogen metabolism under alkali stress Osmotic stress affects plants tissue functions immediately, and excessive Na+ accumulation and high pH value in plant cells cause secondary stresses when plants are subjected to alkali stress (Yang et al., 2007; Guo et al., 2016). In higher plants, some carbohydrates can act as osmoprotectants when plants are subjected to osmotic stress (Hare et al., 1998; Maughan, 2009). The absorption, transmission, and conversion of light energy are essential photosynthetic processes, in which the PS II reaction center plays an important role (Schelvis et al., 1994). To gain more insights into how photosynthesis-related proteins are

Fig. 4. Heatmap of alkaline-stress-induced DAPs. Differentially accumulated patterns in calcium-related, ion-balance, betaine-related, photosystem-related, antioxidative-system, and heat shock proteins.

carbohydrate and energy metabolism (Figs. 2–5, Tables 1 and 2). Additionally, these results corroborate those of Wang et al. (2012b) and Guo et al. (2016) in rice and cotton, respectively. Furthermore, alkali stress enhanced the accumulation of proteins related to oxidoreductase

Table 3 Overview of 40 important protein accumulation situations in young and old cotton leaves. URP, up-regulated proteins; DRP, down-regulated proteins; NCP, nonchanged proteins. Protein type

Calcium-related Ion-balance Betaine-related Photosystem Antioxidative-system Heat shock proteins Sum

Young leaves

Old leaves

Total

Number of URP (percentage)

Number of DRP (percentage)

Number of NCP (percentage)

Number of URP (percentage)

Number of DRP (percentage)

Number of NCP (percentage)

4 (100.00%) 9 (64.29%) 1 (50.00%) 3 (42.86%) 4 (80.00%) 4 (50.00%) 25 (62.50%)

0 (0.00%) 5 (35.71%) 0 (0.00%) 2 (28.57%) 1 (20.00%) 3 (37.50%) 11 (27.50%)

0 0 1 2 0 1 4

1 (25.00%) 11 (78.57%) 2 (100.00%) 2 (28.57%) 3 (60.00%) 2 (25.00%) 21 (52.50%)

3 0 0 3 0 3 9

0 (0.00%) 3 (21.43%) 0 (0.00%) 2 (28.57%) 2 (40.00%) 3 (37.50%) 10 (25.00%)

(0.00%) (0.00%) (50.00%) (28.57%) (0.00%) (12.50%) (10.00%)

375

(75.00%) (0.00%) (0.00%) (42.86%) (0.00%) (37.50%) (22.50%)

4 14 2 7 5 8 40

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Fig. 5. The DAPs involved in the photoreaction system in leaves of cotton. Proteins of the photoreaction system on the left, up-regulation protein in red, downregulation protein in green. Young leaves-Y; Old leaves-O. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

involved in tolerance to alkali stress, we analyzed the DAPs of the photoreaction systems from young and old leaves (Fig. 5). We found that most photoreaction system proteins in young leaves were distinctly up-regulated, compared to old leaves, especially the PS II proteins, suggesting that the enhanced photosynthetic activity resulted from increased photosynthetic electron transport through PS II in young leaves, whereas photosynthetic activity was inhibited in old leaves. A number of key proteins involved in carbon metabolism, such as PRK, Rubisco, PGK, GAPDH, and SBPase, were up-regulated in young leaves, and these proteins were associated with CO2 assimilation, reduction, and ribose regeneration. In young leaves, the up-regulated PRK and Rubisco enhanced the net photosynthetic CO2 assimilation and photorespiratory carbon oxidation (Iqbal et al., 2014). The young leaves might consume increased energy to promote the conversion of glycerate-3P to glyceraldehyde-3P, as Calvin cycle intermediate, by upregulating PGK and GAPDH expression levels (Ge et al., 2015). SBPase is a key regulatory enzyme in the regeneration phase of photosynthetic dark reactions, and, in the present study, up-regulation SBPase induced increases in the regeneration capacity of RuBP and photosynthetic rate, leading to increased photosynthetic carbon assimilation in young leaves (Raines, 2003). Compared with young leaves, carbon metabolism was

Table 4 Summary of the differential response of young vs old leaves of cotton at chlorophyll content and protein functional categories under alkali stress treatment. Differentially accumulated proteins (DAPs). Chlorophyll Protein functional categories

Physiological

Chlorophyll

Proteomic

DAPs

Young leaf/Old leaf Up-regulated

Down-regulated

Chlorophyll a Chlorophyll b Carotenoids Total pigments Carbohydrate metabolism Amino acid metabolism Nitrogen metabolism Photoreaction system Lipid metabolism

Chlorophyll a/b

Energy metabolism Transport and catabolism Signal transduction Cell death proteins

376

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photosynthesis. Thus, this functional division of the same protein family between young and old leaves may indicate active regulation and be of great significance to plants under alkali-stress conditions. In response to alkali stress-related stimuli, the accumulation of ROS, which is a signaling molecule in defense and adaptive responses in plants, is often observed (Yang et al., 2007, 2010). The enhanced ROS generation is followed by increases in antioxidant protein activities in plants (Hu et al., 2012; Wang et al., 2013). CAT, APX, POD, and SOD are key proteins for ROS scavenging in plants, but we determined that there were no significant differences in their levels between control and alkali-stress conditions in both young and old leaves. In this study, GST families had increased expression levels at both developmental stages. Thus, GST might play an important role in responses to alkali-toxicity through the synthesis of molecules required for alkali detoxification in cotton leaves. Heat-shock proteins (Hsps), as a group of molecular chaperones, are responsible for protein folding, assembly, translocation, and degradation and, thus, play crucial roles in maintaining functional conformation of proteins and preventing the aggregation of non-native proteins (Wang et al., 2004). Hsps have essential functions in the reestablishment of cellular homeostasis and in protecting plants against stresses, including drought, salinity, osmotic pressure and heat (Chauhan et al., 2013; Liu and Charng, 2013; Fragkostefanakis et al., 2014). In this study, we identified 8 Hsps, which were differentially accumulated either in young or old leaves, including 2 Hsp90s (HSP90B_1 and HSP90B_2) and 6 Hsp70s (HSPA1_8_1, HSPA1_8_2, HSPA1_8_3, HSPA1_8_4, HSPA4 and HSPA5). The accumulation patterns in two Hsps (HSPA4 and HSPA5) were divergent; therefore, these may serve as factors that result in the down-regulation of the ribosome pathway (ko03010) in young leaves (Fig. 3), because Hsp70 can prevent the aggregation of non-native proteins. The Hsp90 in the young leaves is more sensitive than that in old leaves. Thus, alkali stress is less of a threat to young leaves than to old leaves, owing to Hsp90's genetic buffering role. Indeed, when an organism encounters environmental assaults, Hsp90 has a “buffering” effect that masks genetic variations (Wang et al., 2004). Thus, either Hsp70 or Hsp90 may be responsible for cotton's tolerance to alkali stress as well as for the different responses of the young and old leaves to alkali stress.

weakened in old leaves when the key proteins GAPDH and SBPase were down-regulated under alkali-stress conditions. Accordingly, young leaves probably may enhance their alkali tolerance by increasing photosynthesis and energy consumption. Moreover, carbon metabolism is a core pathway for the synthesis of molecules that are required for alkalistress tolerance in young leaves. Nitrogen metabolism is a basic process of plant physiology, and plant nitrogen assimilation is required for the synthesis and conversion of amino acids through nitrate reduction (Baki et al., 2000; Coruzzi and Bush, 2001). Alkali stress did not influence the expression levels of proteins that are associated with amino acid synthesis in old leaves, whereas it greatly reduced their expression in young leaves. Therefore, alkali stress reduced the nitrogen contents in old and young leaves; however, the reduction in young leaves was greater than that in old leaves. Alkali stress greatly reduced the expression levels of NR and NiR, suggesting that the alkali stress related to the lack of external protons owing to the high pH value might have caused a reduction in NH4+ production in leaves, which destroyed the transmembrane proton gradient that was inhibited by the H+/NO3− symport mechanism (Crawford and Glass, 1998; Wang et al., 2012b). The alkali stress caused GS up-regulation in young leaves but caused its down-regulation in old leaves, whereas GOGAT caused down-regulation in young and old leaves. Thus, alkali stress reduced the ability of GS/GOGAT to assimilate ammonium in leaves; however, the up-regulation of GS reduced the amount of NH4+ by enhancing the assimilation of NH4+ released owing to the increased amino acid catabolism that occurs during alkali stress (Lam et al., 1996). Proline acts as an osmoprotectant when its accumulation is induced by salinity stress in plants (Liu and Zhu, 1998). In our study, P5CS was up-regulated in cotton leaves subjected to alkali stress, and similar up-regulated expression levels of P5CS have been reported by Wang et al. (2012b) in rice and by Chen et al. (2009b) in sunflower. Comparing the tissues, we found that the effect of alkali stress on the nitrogen metabolism in young leaves was greater than in old leaves. 4.3. Analysis of the identified proteins related to alkali tolerance Ion injury, osmotic imbalance and reactive oxygen species (ROS) coupled with alkali stress, severely threaten the growth and development of plants (Shi and Wang, 2005). Ca2+, acting as a second messenger, plays crucial roles in stress signal transduction pathways in higher plants (Liu and Zhu, 1998). CALR and CANX are endoplasmic reticulum proteins that participate in Ca2+ metabolism, and the activation and delay of Ca2+ signaling may influence downstream protein accumulations (Gelebart et al., 2005). CALR-1, CALR-2 and CANX expression levels were up-regulated in young leaves and down-regulated in old leaves, suggesting that young leaves enhance tolerance and alleviate injury, possibly by enhancing the Ca2+ influx by activating Ca2+ channels and increasing Ca2+ concentration. Thus, Ca2+ sensors may be pivotal factors in the physiological and molecular divergence of young and old leaves under alkali stress. Ion balance is an essential strategy for ion detoxification and to establish an osmotic balance (Flowers and Colmer, 2008; Munns and Tester, 2008; Plett and Møller, 2010; Negrão et al., 2017). Plants maintain low Na+ levels in their cytoplasm though, Na+/H+ or Na+/ K+ exchanges, which reduce the unfavorable effects of Na+ under saltor alkali-stress conditions. In this process, NHX, HKT, SOS, and vacuolar H+-ATPase (V-ATPase) proteins play significant roles. Unfortunately, we did not find differentially accumulated NHX, HKT or SOS proteins, perhaps due to a technical limitation. However, we found 14 differentially accumulated V-ATPases, with 6 V-type H+-transporting ATPase, 4 F-type H+-transporting ATPase and 4 PMA. In total, 7 of the 14 V-ATPase proteins were differentially accumulated in young and old leaves. This different accumulation pattern was also observed for betBs (1 out of 2), which serve as essential osmolyte in osmotic regulation, and psbs (3 out of 6), which play key roles in

5. Conclusions We revealed the divergent responses of young and old cotton leaves to alkali stress by using iTRAQ. A total of 4704 high-confident proteins were identified, with 1490 and 563 DAPs in young and old leaves, which indicate more remarkable metabolic flexibility in young leaves than in old leaves. Our results indicated that young and old leaves have distinct protein profiles and mechanisms regulations might be related to the following several factors: first, in response to alkali stress, young leaves maintained stable pigment accumulation but old leaves were strongly damaged. Second, young leaves retained relatively stable carbon metabolism though increased photosynthesis process and energy consumption, whereas carbon metabolism was highly reduced in old leaves. Third, the effect on the nitrogen metabolism of young leaves was stronger than that of old leaves. Fourth, the up-regulated expression levels of the important identified proteins that were related to alkali resistance in young leaves were higher than those in old leaves. To sum up, we identified more differentially abundant proteins than those in previous studies in other plant leaves, and compared the alkali stress-responsive proteins and mechanisms between young and old leaves. These data will increase our understanding of alkali-tolerant mechanisms in higher plants. This study provides a theoretical basis for the excavation of alkaline tolerance proteins of different tissues of cotton and the exploitation and utilization of cotton resource.

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Competing financial interests

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