Comparative adaptive strategies of old and young leaves to alkali-stress in hexaploid wheat

Journal Pre-proof Comparative adaptive strategies of old and young leaves to alkali-stress in hexaploid wheat Chaoxia Xiao, Xiulin Cui, Huiying Lu, Lei Han, Sibei Liu, Yuxin Zheng, Huan Wang, Hao Wang, Chunwu Yang

PII:

S0098-8472(19)31552-7

DOI:

https://doi.org/10.1016/j.envexpbot.2019.103955

Reference:

EEB 103955

To appear in:

Environmental and Experimental Botany

Received Date:

3 October 2019

Revised Date:

20 November 2019

Accepted Date:

21 November 2019

Please cite this article as: Xiao C, Cui X, Lu H, Han L, Liu S, Zheng Y, Wang H, Wang H, Yang C, Comparative adaptive strategies of old and young leaves to alkali-stress in hexaploid wheat, Environmental and Experimental Botany (2019), doi: https://doi.org/10.1016/j.envexpbot.2019.103955

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Comparative adaptive strategies of old and young leaves to alkali-stress in hexaploid wheat Chaoxia Xiao1, Xiulin Cui1, Huiying Lu1, Lei Han, Sibei Liu1, Yuxin Zheng1, Huan Wang2, Hao Wang1, and Chunwu Yang1, * 1

Key Laboratory of Molecular Epigenetics of Ministry of Education, Northeast Normal University, Changchun 130024, China 2

Department of Agronomy, Jilin Agricultural University, Changchun 130118, China

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Highlights

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Chaoxia Xiao: [email protected] Xiulin Cui: [email protected] Huiying Lu: [email protected] Lei Han: [email protected] Sibei Liu: [email protected] Yuxin Zheng: [email protected] Huan Wang: [email protected] Hao Wang: [email protected]

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*Corresponding Author: Dr. Chunwu Yang E-mail: [email protected] Key laboratory of Molecular Epigenetics of Ministry of Education (MOE), Northeast Normal University, Changchun 130024, China

Proteomics data are based on newly released reference genome of common wheat.

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Under alkali stress, old leaves may programmatically degrade proteins and ribosomes through the ubiquitin-proteasome pathway to generate amino acids for osmotic adjustment. Alkali stress influences the translation of homoeologous genes in hexaploid wheat, resulting in imbalanced translation of A, B, and D homoeologous genes in some gene triads.

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Abstract

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Alkali stress is an important limiting factor for agricultural production. The aim of this study was to investigate whether old and young leaves have different biochemical responses to alkali stress in hexaploid wheat, and to provide molecular dissection of these differential responses on the basis of proteomic profiling. Under alkali stress, hexaploid wheat accumulated higher concentrations of Na+ in older leaves to protect young leaves. Young leaves accumulated amino acids, carbohydrates, and dehydrin proteins to relieve Na+ toxicity. Under alkali stress, 14-3-3 protein abundance was enhanced in old leaves to accelerate Na+ compartmentation in the vacuole. Abundances of 27 ribosomal proteins were significantly decreased in old leaves but not in young leaves, and many proteinases and ribonucleases also were significantly upregulated by alkali stress in old leaves. Interestingly, one E3 ubiquitin-protein ligase, one ubiquitin protein, and one proteasome protein were simultaneously upregulated in old leaves but not in young leaves. We propose that during the response to alkali stress, 1

old leaves may programmatically degrade ribosomes through the ubiquitin-proteasome pathway to generate amino acids, which will increase tissue tolerance. Alkali stress induced remarkable enhancement of almost all free amino acids and ribose contents in old leaves, thus supporting this hypothesis. In addition, we observed that, for some salinity-tolerant protein-triads, the A, B, and D homoeologous proteins showed different response to alkali stress. This suggests that alkali stress may influence the translation of homoeologous genes, resulting in imbalanced translation of A, B, and D homoeologous genes in some gene triads. Keywords：

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Ribosome Protein degradation Amino acid Carbohydrate Homoeologous protein Na+ toxicity 1. Introduction

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Currently, 1.5 × 109 ha land are cultivated across the world. Of these cultivated lands, about 0.34 × 109 ha (23%) are saline (caused by NaCl) and another 0.56 × 109 ha (37%) are sodic (caused by NaHCO3 and Na2CO3) (Läuchli and Lüttge, 2002). Alkali stress (NaHCO3 and Na2CO3) has been considered as an important environmental factor in limiting agricultural production, and has led to severe problems in some areas. For example, in northeast China, more than 70% of grassland is alkaline (Kawanabe and Zhu, 1991). Salt tolerance has been investigated for almost 40 years (Abdelraheem et al., 2019; Ganie et al., 2019; Han et al., 2019; Vaishnav et al., 2019; Wang and Xia, 2018), but few studies have focused on the alkali tolerance of plants. Recent alkali tolerance studies

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have focused on transgenesis (Wang et al. 2016; He et al. 2017), physiological response and gene expression (Zhang et al., 2013; Jia et al. 2019), metabolic response (Guo et al. 2016), and proteomic profiling (Zhang et al., 2016a; Zhao et al. 2019).

Osmotic adjustment is essential for plant survival under salinity or alkali stress. Accumulating Na and Cl− in the vacuole largely contributes to the osmotic potential of plant cells under salinity stress or alkali stress, but minimal organic solutes play osmotic roles only in the cytoplasm (Munns et al., 2016). Another adaptive mechanism is tissue tolerance, defined as the capacity of organs or tissues to relieve ion toxicity in the presence of Na+ and Cl− concentrations elevated as high as necessary for osmotic adjustment (Munns et al., 2016). Young and old leaves have different tissue tolerance mechanisms because old leaves have a larger vacuole, but young leaves only have miniature vacuoles (Munns and Tester, 2008). Under salt or alkali stress, to protect young and mature leaves, plants usually accumulate toxic ions (e.g. Na+ and Cl−) into the vacuoles of old leaves (Akram et al., 2006; Ashraf and O'Leary, 1997; Hajlaoui et al., 2010; Nakamura et al., 1996; Vera-Estrella et al., 2005; Wang et al., 2012b; Yasar et al., 2006). Continuous production of new leaves and increased survival of older leaves are necessary for maintaining the survival and growth rate of plants under salt or alkali stress. Increased survival of older leaves can relieve the ion toxicity of the whole plant, while the increased survival of young leaves allows continuous production of new leaves to supply more carbohydrates for roots and old leaves (Munns and Tester, 2008). These differences of construction and function between young and old leaves suggest that they have different adaptive metabolisms to salt or alkali stress. However, relatively little attention has been given to comparative adaptive strategies of old and young tissues to salt or alkali stress. In this area, most attention has focused on salt stress (Akram et al., 2006; Ashraf and O'Leary, 1997; Hajlaoui et al., 2010; Nakamura et al., 1996; Vera-Estrella et al., 2005; Yasar et al., 2006), with few studies focused on alkali stress.

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2. Materials and methods

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Wang et al. (2012b) report that alkali stress produces different effects on gene expression of old and young leaves in rice plants, and Guo et al. (2016) report that alkali stress has different effects on metabolite accumulation in old and young leaves in cotton plants. However, these two studies do not provide molecular insights into alkali tolerance. Wheat is one of the most important crops in the world, and provides about one-fifth of the calories consumed by humans (Dubcovsky and Dvorak, 2007). Of the wheat crop, 95% is hexaploid common wheat (Triticum aestivum L., genome BBAADD), whereas the remaining 5% is the ancestor of hexaploid wheat, durum wheat (Triticum turgidum, genome BBAA). The evolutionary success of the hexaploid wheat may be attributable to its higher ploidy level and complex genome composition. The genome of hexaploid wheat consists of A, B, and D subgenomes, and extensive interactions among the three subgenomes lead to silencing of homoeolog-specific expression or even loss of DNA fragments and the alteration of chromosome structure (Akhunov et al. 2013; Zhang et al. 2017; Zhang et al. 2014). Salinity stress strongly influences interactions between the BBAA genome and the DD genome, causing expression pattern alterations of homoeologs in hexaploid wheat (Zhang et al., 2016b). However, it is unclear whether salinity or alkali stress have different effects on the abundances of protein encoded by the A, B, and D homoeologous genes of a gene triad in hexaploid wheat. In the present work, we aimed to address following three questions: 1. Do old and young leaves of hexaploid wheat employ different physiological mechanisms to resist alkali stress? 2. Do old and young leaves show different proteomic response to alkali stress, and can we provide molecular dissection of differential physiological responses of old and young leaves to alkali stress? 3. Do the three subgenomes play different roles in the proteomic response of hexaploid wheat to alkali stress? To address these questions, we measured Na+, K+, and 38 compatible solute contents of old and young leaves in hexaploid wheat under alkali stress. We also conducted a proteomics analysis using a high-quality hexaploid wheat reference genome (iwgsc_refseqv1.0) with precise homoeolog information, allowing us to distinguish A, B, and D homoeologs for each gene triad (Alaux et al., 2018; Ramírez-González et al., 2018).

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2.1. Plant material and stress treatment

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‘Bobwhite’, a hexaploid wheat cultivar (Triticum aestivum L., genome BBAADD), was used in this research. This cultivar is widely used in transgenic experiments and molecular biology research. The seeds were kindly provided by Dr. Daolin Fu (Shandong Agricultural University, China). Seeds of ‘Bobwhite’ were sown in pots containing thoroughly washed sand. Each pot contained 10 seedlings. All pots were placed in a greenhouse with a thermoperiod of 22–25/14–17 °C and a 16/8 h day/night photoperiod. The pots were watered with half-strength Hoagland nutrient solution for 25 days. After this treatment, the pots were treated with alkali stress solution (NaHCO3/Na2CO3, 9:1; 100 mM, pH 8.7) containing nutrient components of half-strength Hoagland nutrient solution. Control pots were watered with half-strength Hoagland nutrient solution. Stress treatment duration was 4 days. 2.2. Biochemical analysis Fresh old (second leaf from bottom) and young (newly emerged) leaves were freeze-dried for biochemical analyses. Ten leaves from five individuals were pooled to form a biological replicate, with three replicates for each treatment and each tissue. Free amino acids and sugars of the dried samples were measured using the methods of Zhao et al. (2017). Briefly, the free amino acids and sugars were extracted with distilled water at 3

50 °C, and the extracted solutions were further treated and loaded into a high-performance liquid chromatograph line to a triple quadrupole mass spectrometer (API3200MD, AB SCIEX, USA) to determine the contents of these solutes. Dried samples of old and young leaves were digested three times in 65% HNO3 at 120 °C, and their Na+ and K+ contents were measured by an atomic absorption spectrophotometer (TAS990super, PERSEE, China). Percentage contribution of each compatible solute to total molarity is

calculated using following formula: percentage contribution of a given solute = its molarity (μmol g1 DW) × 100%/total molarity, where total molarity is the sum of the molarity of all 38 detected compatible solutes. 2.3. Protein extraction and alkylation

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Label-free quantification proteomic analysis was conducted using the workflow of Han et al.(2019). Old and young leaves of five individuals for each treatment were pooled as a biological replicate. Each treatment included four biological replicates. Proteins were extracted and precipitated from old and young leaves using TCA/acetone solution (10% TCA in acetone). The pellet was dissolved in buffer A (8 M urea, 4% Chaps, 30 mM HEPES, 2 mM Na2EDTA, 10 mM DTT, and 1 mM PMSF; pH 8.2. Then, 15.8 μl of 200 mM DTT was added into 0.3 ml of the supernatant. After incubation for 1 h at 56 °C, 35 μl of 1 M iodoacetamide was added to the solution. Finally, the pellet was dissolved in 50 mM NH4HCO3. The protein was quantified using the Bradford method (Bradford, 1976).

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2.4. Protein digestion and LC-MS-MS analysis

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Trypsin (1 μg) was used to digest 40 μg protein into peptides for 16 h at 37 °C. Thermo Scientific Pierce C18 Pipette Tips (production ID 87784) were then used to purify the peptides. The purified peptides were loaded onto an EASY-nLC 1200 system equipped with a Q-Exactive mass spectrometer and a nano-electrospray ion source (Thermo Scientific, Germany). Label-free proteomic analysis was conducted according to the protocol of Thermo Scientific. The mass spectrometer and EASY-nLC 1200 system were set following the parameters of previous work (Han et al., 2019).

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2.5. Label-free quantification For label-free quantification, Proteome Discoverer software 2.2 (Thermo Scientific, USA) was used to analyze the MS-MS raw data generated by Xcalibur software against the common wheat reference genome (iwgsc_refseqv1.0). The reference genome sequences were downloaded at http://www.wheatgenome.org on September 13, 2018. Proteins were identified using unique peptides with false detection rate < 0.05. Differentially accumulated proteins (DAPs) between treatments were identified by ANOVA analysis (background-based) using Proteome Discoverer version 2.2 according to the manufacturer’s instructions, and the generated p-values were adjusted by the BenjaminiHochberg method. DAP was defined as a fold-change > 2 and an adjusted P value < 0.05 between alkali stress and control conditions. 2.6. qRT-PCR

Old and young leaves of five individual seedlings for each treatment were pooled as a biological replicate, with four biological replicates. qRT-PCR was preformed according to conventional methods (Han et al., 2019). Real-time PCR analysis was carried out using StepOnePlus system and SYBR Green Real-time PCR Master. Gene-specific primers are listed in Table S1. Actin and RLI were 4

used as internal control genes (Lv et al., 2019). Gene expression data were normalized using the △△Ct method (Livak and Schmittgen, 2001). 2.7. Assignment of genes in the wheat reference genome and subgenomes

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According to gene ID information for the hexaploid wheat reference genome (iwgsc_refseqv1.0), each gene was assigned to the A or B or D subgenome (Alaux et al., 2018; Ramírez-González et al., 2018). For example, in “TraesCS3B01G207000.1”, “3B” means that this gene is located on the 3B chromosome; G indicates "gene"; 207000 represents an increasing number in steps of “100” along the respective chromosome arm; .1 indicates splice variant "1". Triads were defined according to Ramírez-González et al. (2018). A homoeolog triad is exclusively composed of one A subgenome copy, one B subgenome copy, and one D subgenome copy (Ramírez-González et al., 2018). Based on the identification of Ramírez-González et al. (2018), 17,400 syntenic and 1074 nonsyntenic triads were identified, and all identified triads had a 1:1:1 correspondence across the three homoeologous subgenomes (total of 18,474 triads or 55,422 genes). 2.8. Statistical analysis

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The experimental design was a randomized complete block design. All data were from 3-4 biological replicates. Statistical analysis of biochemical data was performed using SPSS version 16.0 (SPSS, Chicago, IL, USA). The statistical significance of physiological measurement was determined by the t-test at the 0.05 level. Statistical tests for label-free quantification were performed using Proteome Discoverer version 2.2 based on the ANOVA (background-based) method (adjusted P value < 0.05).

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3. Results

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3.1. Physiological response

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Na+ concentration was enhanced under alkali stress in both old and young leaves, with higher concentrations in old leaves than in young leaves (Figure 1). We did not detect methionine in old leaves, but detected trace amounts in young leaves. In young leaves, alkali stress enhanced the contents of three of the other 19 free amino acids (proline, asparagine, and histidine) by 30%–70%; however, in old leaves, alkali stress increased the contents of these four amino acids by 120%–560% (Figure 2). Alkali stress increased the contents of almost all free amino acids in old leaves of hexaploid wheat (Figure 2). Alkali stress increased contents of erythrose, fructose, glucose, mannose, sucrose, lactose, xylitol, maltose, and total carbohydrate in young leaves, and enhanced contents of sorbitol and mannitol, xylitol, fucose, and ribose in old leaves (Figure 3). To survey the contribution of each solute to osmotic adjustment, we calculated percentage contribution of each solute molarity to total molarity (Table 1). Under alkali stress, percentage contributions of most amino acids to total molarity decreased in young leaves but increased in old leaves. Eight out of 14 carbohydrates showed stress-increased percentage contributions to total molarity (Table 1). Under alkali stress, percentage contribution of fructose (11.17%), glucose (13.38%), and sucrose (42.76%) was greater than that of any other solute in young leaves (Table 1). In stressed old leaves, alanine (13.12%), sucrose (15.18%), and fructose (7.74%) displayed greater percentage contributions to total molarity than the other solutes (Table 1). Polyols had small roles in osmotic adjustment. 3.2. Differentially accumulated proteins 5

Under our experimental conditions, we detected 3787 master proteins (Table S2). According to protein ID information, these master proteins consisted of 1677 A subgenome proteins (AP), 1140 B subgenome proteins (BP), and 935 D subgenome proteins (DP). The reference genome (iwgsc_refseqv1.0) of hexaploid wheat includes 36,302 A-subgenome genes, 36,737 B-subgenome genes, and 35,021 D-subgenomes genes. Although the three subgenomes consist of almost equal numbers of genes, more A subgenome proteins were detected than B or D subgenome proteins. In old leaves, we found 457 differentially accumulated proteins (DAPs) under control and stress conditions, including 279 DAPs upregulated and 178 DAPs downregulated (Figure 4 and Table S3). However, 119 upregulated DAPs and 170 downregulated DAPs were found in young leaves. Twenty downregulated DAPs and 28 upregulated DAPs were shared by old and young leaves (Figure 4 and Table S3). Interestingly, 26 proteins were upregulated in old leaves but downregulated in young leaves (Figure 4 and Table S3). Ten proteins were downregulated in old leaves but upregulated in young leaves (Figure 4 and Table S3). 3.3. Alkali tolerance proteins

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Five peroxidase proteins (four APs + one BP) were upregulated by alkali stress in old leaves but not in young leaves, another four peroxidase proteins (two DPs + one BP + one AP) were upregulated only in young leaves, and only one peroxidase protein (TraesCS7D01G461500.1) was upregulated in both leaf tissues (Table 2). In old leaves, alkali stress enhanced the abundances of one superoxide dismutase [Cu-Zn], one peroxidase (TraesCS6A01G047200.1), and three glutathione S-transferase proteins (TraesCS1A01G078800.1, TraesCS5A01G502300.1, and TraesCS3A01G302100.1); however, these enzymes were not upregulated in young leaves (Table 2). Abundances of one ascorbate peroxidase (TraesCS2D01G080000.1) and two dehydrin proteins (TraesCS7D01G549900.1 and TraesCS6D01G333000.1) were enhanced in young leaves but not in old leaves. Under alkali stress, a 14-3-3 protein (TraesCS4B01G159900.1) also was upregulated in old leaves but not in young leaves. A dehydrin protein (TraesCS6D01G234700.1) was downregulated

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in both leaf tissues, and one protein phosphatase 2c protein (TraesCS2D01G426600.1) was upregulated in both leaf tissues (Table 2). 3.4. Degradation of proteins and ribosomes

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We identified 43 differentially accumulated ribosomal proteins under control and alkali stress conditions (Table 3). Of these ribosomal proteins, 26 were greatly downregulated by alkali stress in old leaves but not in young leaves, six ribosomal proteins were downregulated in both leaf tissues (Table 3). Three cysteine proteinases (TraesCS2D01G272400.1, TraesCS2A01G273100.1, and TraesCS5A01G326100.3), a cysteine protease (TraesCS1B01G338500.1), a nuclease S1 (TraesCS2D01G507900.1), and two ribonucleases (TraesCS3D01G392300.1 and TraesCS2B01G182900.1) were upregulated in old leaves but not in young leaves (Table 4). Another two ribonucleases (TraesCS6D01G320200.1 and TraesCS6A01G339600.1) were upregulated in both leaf tissues, with greater upregulation in old than in young leaves (Table 4). In old leaves, alkali stress increased the abundance of the E3 ubiquitin-protein ligase CHFR (TraesCS3D01G079000.2) by 60%, but this was not statistically significant (Table 4). Proteasome subunit beta (TraesCS6A01G145800.1) and a ubiquitin family protein (TraesCS4B01G325900.2) were upregulated in old leaves but not in young leaves. 3.5. Homoeologous proteins Each gene of hexaploid wheat theoretically consists of three homoeologs (A, B, and D) (triad). However, according to reference genome information for hexaploid wheat, only about 50% of high6

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confidence genes are assigned to a triad, with a total of 18,474 triads (55,422 genes) (RamírezGonzález et al., 2018). Here, we defined protein-triad as a set of three homoeologous proteins, representing translation productions of the three homoeologous genes (A, B, and D) of a gene triad. We detected only 40 protein-triads that contained entire A, B, and D homoeologous proteins (Figure 5). Rectangular coordinate plots were drawn for all of 40 protein-triads with x = fold change of homoeologous protein of A subgenome (FCHP-A) and y = FCHP-B, as well as x = FCHP-A and y = FCHP-D and x = FCHP-B and y = FCHP-D (Figures 5a-c). The icons aggregating to a diagonal line indicated that the protein-triads showed small differences between subgenomes in FCHP. In most of the 40 protein-triads, A, B, and D homoeologous proteins showed similar response patterns to alkali stress (Figures 5A-C). Results for a methyl-CpG binding domain protein-triad and a dehydrin proteintriad are particularly interesting. For the methyl-CpG binding domain protein-triad (Figure 5D), fold changes (stress/control) of the three homoeologous proteins were similar in young leaves, but alkali stress only revealed a small effect on the abundances of the A and D homoeologous proteins in old leaves but significantly decreased the abundance of the B homoeologous protein. For the dehydrin protein-triad, in young leaves, alkali stress slightly upregulated the B homoeologous protein, greatly downregulated the D homoeologous protein, and did not influence the abundance of the A homoeologous protein; however, in old leaves, alkali stress slightly increased the abundances of the A and B homoeologous proteins (no statistical significance), and mightily decreased the abundance of the D homoeologous protein (Figure 5E). 3.6. qRT-PCR

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For ten out of 14 selected genes tested, the fold-change values from label-free analysis were similar to those from qRT-PCR data (Table S1); therefore, the results of label-free quantification analysis were reliable. 4. Discussion

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4.1. Osmotic adjustment, tissue tolerance, and antioxidant response

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Salt stress limits growth of plants by osmotic stress and ion toxicity (Munns 2002). Comparison of alkali stress with salt stress reveals an added high-pH injury. Osmotic stress has a faster effect on growth than ion stress, especially at low and moderate salinity levels. The ion-specific toxicity of salt stress or alkali stress is present only when toxic ions accumulate to toxic concentrations, which may take several days or even several weeks (Munns and Tester, 2008). Osmotic stress caused by salinity or alkali stress causes a faster and stronger limitation on cell expansion of young leaves than that of old leaves (Munns and Tester, 2008; Parida and Das, 2005). The osmotic adjustment capacity of young leaves may be important for the growth rate of hexaploid wheat under alkali stress. Accumulating Na+ and Cl- in the vacuole makes a dominant contribution to the enhancement of osmotic pressure, with less energy demand than the synthesis of organic solutes (Munns et al., 2016). Once the vacuole storage threshold of a leaf tissue is reached, continued entry of these ions into the cytosol will lead to injury to biomacromolecules and organelles. Therefore, relieving Na+ toxicity in the cytosol is critical for plant survival of high-salinity conditions (Munns et al., 2016). To balance the osmotic stress from Na+ in vacuoles as well as to protect organelles or biomacromolecules, plants will synthesize compatible solutes or protective proteins in the cytosol. Protective proteins include dehydrin and LEA proteins that are composed of tandem hydrophilic amino acids (highly hydrophilic structure) and likely play important roles in the prevention of cytosol dehydration and protein aggregation under salinity or drought stress (Rorat, 2006). High pH during alkali stress severely inhibits the uptake of K+ and enhances accumulation of Na+ in plants (Guo et al. 2017; Wang et al. 2011; Wang et al. 2012a; Yang et al. 2008). In the present 7

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work, we observed high concentrations of Na+ in old leaves as well as in young leaves under alkali stress, but with higher concentrations in old leaves than in young leaves. Under alkali stress, old leaves accumulated higher concentrations of Na+ to protect the young leaves (Figure 6). Nevertheless, partial Na+ still entered young leaves of wheat under alkali stress. As young leaves lack large vacuoles, free Na+ concentration in their cytosol may be extremely high under alkali stress; therefore, the functional protection of biomacromolecules and organelles from Na+ injury is crucial for maintaining the normal metabolism of young leaves (Figure 6). We found that the accumulation of many amino acids and carbohydrates was induced by alkali stress. Under alkali stress, three carbohydrates (fructose, glucose, and sucrose) were dominant compatible solutes in young leaves (Table 1). However, in stressed old leaves, the contribution of amino acids to osmotic adjustment was significantly enhanced, and alanine became one of the dominant compatible solutes (Table 1). Polyols played a small role in osmotic adjustment of alkali-stressed wheat. Our results also showed that, under alkali stress, accumulation of two dehydrin proteins (TraesCS7D01G549900.1 and TraesCS6D01G333000.1) was induced in young leaves but not in old leaves, suggesting that the dehydrin proteins may play important roles in cytosol dehydration resistance and the protection of cytosol proteins in young wheat leaves under alkali stress. Another important function of dehydrin proteins is formation of intracellular glasses by interaction with carbohydrates. The intracellular glasses can slow molecular mobility of water and ions (Buitink and Leprince 2008), which may limit the movement of Na+ and Cl- in the cytoplasm to mitigate their toxicity. Combination of increasing carbohydrate contents and enhancing dehydrin protein abundance may result in large accumulation of intracellular glasses in wheat young leaves under alkali stress, alleviating ion toxicity (Figure 6). The salt overly sensitive (SOS) pathway is a conserved salt tolerance pathway in higher plants, which regulates Na+ compartmentation and excretion under salt stress (Yang et al., 2019; Zhu, 2003). The SOS pathway is composed of SOS3 (a calcium-binding protein), the SOS2 protein kinase, a vacuolar Na+/H+ antiporter (NHX), and a plasma membrane Na+/H+ antiporter (SOS1) (Zhu, 2003). NHX mediates Na+ compartmentation and SOS1 functions in Na+ excretion. The SOS2-SOS3 complex activates SOS1 and NHX proteins through phosphorylation. In Arabidopsis plants, a 14-33 protein was demonstrated to bind Ca2+ and modulate the SOS pathway (Yang et al., 2019). In wheat plants, under alkali stress, a 14-3-3 protein (TraesCS4B01G159900.1) was greatly upregulated in old leaves but not in young leaves (Table 2). The high abundance of 14-3-3 protein likely promotes Na+ compartmentation in the vacuole of old leaves through the SOS pathway. Taken together, under alkali stress, young wheat leaves enhance their dehydrin abundance to protect against cytoplasmic dehydration and Na+ injury of biomacromolecules, and old leaves increase their 14-3-3 protein abundance to promote Na+ compartmentation in the vacuole (Figure 6). We found that many of the peroxidase proteins and glutathione S-transferase proteins of the antioxidant response were upregulated either in old leaves or in young leaves, but different family members were employed in the two leaf tissues. In old leaves, four out of five upregulated peroxidase proteins were from the A subgenome and all three upregulated glutathione S-transferase proteins were from the A subgenome. In young leaves, only one out of four upregulated peroxidase proteins was from the A subgenome. These results indicated that, in old wheat leaves, the A subgenome may play a more important role in the antioxidant response than the B and D subgenomes. 4.2. Degradation of proteins and ribosomes We observed that abundances of 27 ribosomal proteins were significantly decreased in old leaves but not in young leaves, and many proteinases and ribonucleases were also significantly upregulated in old leaves (Tables 3-4). This was not consistent with findings in Medicago plants (Long et al., 2019) in which many ribosomal proteins were significantly upregulated under alkali stress. This suggests that alkali tolerance mechanisms of plants are diverse. In Medicago plants, alkali stress may enhance 8

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assembly rate of ribosomes to increase the synthesis of alkali-responsive proteins such as heat shock protein, late embryogenesis abundant (LEA) protein, and H+-ATPase (Long et al., 2019). Here, we propose the hypothesis that in order to alleviate cytoplasmic Na+ toxicity under alkali stress, ribosomes and some proteins may be programmatically degraded in old leaves but not in young leaves (Figure 6). Our physiological analysis provided two pieces of evidence to support this hypothesis. Response of amino acids to alkali stress is the first evidence. In young leaves of hexaploid wheat, alkali stress only slightly enhanced the contents of four free amino acids (proline, asparagine, glutamine, and histidine). In old leaves of hexaploid wheat, alkali stress greatly increased the contents of almost all free amino acids. If contents of only a few free amino acids were increased, the accumulation may have come from the conversion of other amino acids; since contents of almost all free amino acids were enhanced, the accumulation was most likely the result of degradation of proteins. The second evidence is provided by the observation that ribose content and the abundance of two ribonucleases were significantly enhanced in old leaves but not in young leaves (Figure 3 and Table 4). Increased ribose content in old leaves may be generated from rRNA degradation by the ribonucleases. In old leaves of hexaploid wheat, degradation of ribosomal proteins was also accompanied by the upregulation of many proteinases, which revealed that the ribosome degradation in old leaves may be a precisely controlled programmed process. Interestingly, in wheat plants, an E3 ubiquitin-protein ligase, a ubiquitin protein, and a proteasome protein were simultaneously upregulated in old leaves but not in young leaves. We propose that the degradation of ribosomes in old alkali-stressed wheat leaves may be mediated by the ubiquitin-proteasome pathway (Figure 6). Ribosomes are composed of ribosomal proteins and rRNA. Ribosome degradation in old leaves will produce high concentrations of amino acids and ribose, which can be used to increase tissue tolerance and even translocated to young or mature leaves to enhance their tissue tolerance (Figure 6).

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4.3. Abundance changes in homoeologous proteins under alkali stress

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The genome of hexaploid wheat consists of A, B, and D subgenomes. In hexaploid wheat, interactions between the BBAA genome and DD genome may lead to extensive expression partitioning and function diversification of homoeologs, which provides greater genome and physiological plasticity and enhances stress tolerance (Akhunov et al., 2013; Akhunova et al., 2010; Bottley et al., 2006; Dubcovsky and Dvorak, 2007; Yang et al., 2014). As an allohexaploid, each gene of wheat theoretically consists of three homoeologs (A, B, and D), called a homoeolog triad. Three homoeologs of a triad are expected to share similar expression patterns (Zhang et al., 2016). Transcriptional data cross 15 tissues and multiple growth stages indicated that in 85% of gene triads the three homoeologs were simultaneously expressed, and that 70% of gene triads showed balanced expression of A, B, and D homoeologs (Ramírez-González et al., 2018). It was expected that, for most triads, the three homoeologs would produce a similar abundance of proteins. Interestingly, among the 3787 proteins detected, we found only 40 protein-triads (120 homoeologous proteins) that contained the entire three homoeologus proteins (A, B, and D). It is likely that, for most gene triads, only one or two homoeologs are translated into protein. There may be a translation preference for one of the three subgenomes. Given that throughput of current proteomics technology is limited, only proteins with high abundance and moderate abundance can be detected. We caution that absence of a homoeologous protein in proteomic profiling may be due to its low abundance. Our data at least partially demonstrates that, for most gene triads, translation of A, B, and D homoeologs was severely unbalanced. We hope that this hypothesis will be validated by next-generation proteomic technology with higher throughput in the future. We compared the response of the three homoeologous proteins to alkali stress in protein-triads that contained entire A, B, and D homoeologous proteins. It has been reported that stress conditions have different effects on the expression of the three homoeologous genes of a gene triad (Liu et al., 2015; Zhang et al., 2016). Stressful conditions may have triggered sub- or neofunctionalization of 9

homoeologs during wheat evolution (Zhang et al., 2016). We expected that stress conditions would have different effects on the protein abundances of the A, B, and D homoeologs of the wheat proteintriads. Indeed, in some protein-triads, such as the methyl-CpG binding protein-triad (core components involved in DNA methylation) and dehydrin protein-triad, we observed that the A, B, and D homoeologous proteins showed different response to alkali stress (Figure 5). We propose that alkali stress may shock the homoeologous translation, and even influence the post-translational modification of homoeologous proteins. This should be investigated in the future. 5. Conclusion

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Young leaves and old leaves of hexaploid wheat employ different mechanisms to cope with alkali stress. Young leaves accumulated amino acids, carbohydrates, and dehydrin proteins to relieve Na+ toxicity. Old leaves may programmatically degrade ribosomes through the ubiquitin-proteasome pathway to generate amino acids to increase tissue tolerance. Additionally, alkali stress influences the translation of homoeologous genes in hexaploid wheat, resulting in imbalanced translation of A, B, and D homoeologous genes in some gene triads. All data of this manuscript are original, and have not been considered for publication elsewhere in any form, and all authors have contributed substantially to this work. All authors have read this

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revision, and agreed to this submission.

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Conflicts of interest The authors have declared that no competing interests exist.

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Author Contributions Experiment design: CY; performing experiments: CX, XC, HL; LH, SL, YZ, and HW2 (Hao Wang); data analysis: CX, XC, HL; LH, SL, YZ, and HW1 (Huan Wang); manuscript writing: CY, XC, and HW1. Accession number The mass spectrometry proteomics data were deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository (Ma et al. 2019) with accession no. IPX0001874000/PXD016354.

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (Nos. 31971762, 31670218 and 31802114); the Fundamental Research Funds for the Central Universities (No. 2412019FZ026); and the China Postdoctoral Science Foundation (Nos. 2017M610197 and 2018T110262).

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Figure captions

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Figure 1 Effects of alkali stress on Na+ and K+ contents in old and young leaves of hexaploid wheat plants. Wheat plants (25 days old) were treated with alkali stress (NaHCO3/Na2CO3, 9:1; 100 mM, pH 8.7) for 4 days. Ten leaves from five individuals were pooled to form a biological replicate, with three replicates for each treatment and each tissue. Values are means (± S.D.) of three replicates. * Indicates significant difference between control and stress conditions within the same tissue at the 0.05 level according to t-test.

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Figure 2 Effects of alkali stress on free amino acid contents in old and young leaves of hexaploid wheat plants. Wheat plants (25 days old) were treated with alkali stress (NaHCO3/Na2CO3, 9:1; 100 mM, pH 8.7) for 4 days. Ten leaves from five individuals were pooled to form a biological replicate, with three replicates for each treatment and each tissue. Values are means (± S.D.) of three replicates. * Indicates significant difference between control and stress conditions within the same tissue at the 0.05 level according to t-test.

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Figure 3 Effects of alkali stress on carbohydrate contents in old and young leaves of hexaploid wheat plants. Wheat plants (25 days old) were treated with alkali stress (NaHCO3/Na2CO3, 9:1; 100 mM, pH 8.7) for 4 days. Each treatment and each tissue had three biological replicates. Values are means (± S.D.) of three replicates. * Indicates significant difference between control and stress conditions within the same tissue at the 0.05 level according to t-test.

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Figure 4 Venn diagram showing number of differentially accumulated proteins under control and alkali stress conditions in old and young leaves of hexaploid wheat. Wheat plants (25 days old) were treated with alkali stress (NaHCO3/Na2CO3, 9:1; 100 mM, pH 8.7) for 4 days. Each treatment and each tissue had four biological replicates, and each biological replicate was a pool of five plants.

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Figure 5 Effects of alkali stress on abundance of homoeologous proteins. Protein-triads containing entire A, B, and D homoeologous proteins are shown. (a–c) Rectangular coordinate plot drawn with x = fold change of homoeologous protein of A subgenome ( FCHP-A) and y = FCHP-B, as well as x = FCHP-A and y = FCHP-D and x = FCHP-B and y = FCHP-D. (d-e) Fold changes of homoeologous protein abundance of two typical protein-triads, methyl-CpG binding domain containing protein (d) and dehydrin (e). * Indicates fold change reached a significant level (adjusted P value < 0.05). Wheat plants (25 days old) were treated with alkali stress (NaHCO3: Na2CO3 = 9:1, 100 mM, pH 8.7) for four days. Each treatment and each tissue had four biological replicates, and each biological replicate was a pool of tissues from five plants

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Figure 6. Comparative adaptive strategies of old and young leaves to alkali stress in hexaploid wheat plants.

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Old leaf（%）

Control

Stress

Control

Stress

Glycine

0.36

0.29

0.82

0.46

Alanine

6.89

3.81

11.59

13.12

Serine

1.53

0.87

3.74

6.84

Proline

0.81

0.63

1.07

4.43

Valine

1.07

0.63

1.77

5.39

Threonine

1.21

0.74

2.25

3.39

Cysteine

1.17

0.77

Isoleucine

0.58

0.33

Asparagine

2.37

2.39

Aspartic acid

1.68

0.78

Glutamine

0.40

0.30

Glutamic acid

0.42

Methionine

0.00

Histidine

0.31

Phenylalanine

0.71

1.04

1.02

2.95

1.73

4.95

3.06

2.21

0.84

5.12

0.29

1.22

2.12

0.01

0.00

0.00

0.30

0.78

1.56

0.33

1.33

3.92

lP

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0.63

0.78

0.44

1.11

1.57

0.45

0.26

0.45

1.32

1.05

0.57

1.20

1.45

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Arginine

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Young leaf （%）

Tryptophan Lysine

0.67

0.33

0.76

1.27

Leucine

0.97

0.47

1.35

3.33

Erythrose

6.02

5.63

2.65

1.46

Fructose

10.64

11.17

13.42

7.74

Xylose

2.68

1.06

0.96

0.42

Rhamnose

0.24

0.12

0.10

0.13

Glucose

7.27

13.38

4.40

3.34

Galactose

2.02

1.47

2.14

1.24

Mannose

4.43

5.06

4.27

2.66

Fucose

0.07

0.04

0.04

0.15

Sucrose

40.26

42.76

33.50

15.18

Lactose

0.08

0.26

0.03

0.00

Free carbohydrates

Tyrosine

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Free amino acids

Table 1 Percentage contribution of each compatible solute to total molarity. Percentage contribution is calculated using following formula: percentage contribution of a given solute = its molarity (μmol g-1 DW) × 100%/total molarity, where total molarity is the sum of the molarity of all 38 detected compatible solutes. Wheat plants (25 days old) were treated with alkali stress (NaHCO3/Na2CO3, 9:1; 100 mM, pH 8.7) for 4 days.

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0.90

0.70

0.61

0.26

Stachyose

0.11

1.96

0.11

0.10

Ribose

0.07

0.05

0.01

0.04

Maltose

0.74

1.15

0.24

0.00

Pinitol

0.11

0.09

0.15

0.20

Inositol

0.86

0.50

0.59

0.52

Xylitol

0.01

0.00

0.00

0.01

Sorbitol & Mannitol

0.03

0.03

0.04

0.11

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Polyols

Melitose

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Table 2 Effects of alkali stress on abundances of salinity tolerance proteins in old and young leaves of hexaploid wheat plants. GST, glutathione-S-transferase; Cu-Zn-SOD, Cu-Zn-superoxide dismutase; PP2C, protein phosphatase 2c. Wheat plants (25 days old) were treated with alkali stress (NaHCO3/Na2CO3, 9:1; 100 mM, pH 8.7) for 4 days. Each treatment and each tissue had four biological replicates, and each biological replicate was a pool of tissues from five plants. Gene ID

Fold change

Adj. P Value

Fold change

Adj. P Value

14-3-3 protein

TraesCS4B01G159900.1

0.746

0.846

6.973

0.014

Ascorbate peroxidase

TraesCS2D01G080000.1

2.027

0.000

1.353

0.989

Asparagine synthetase

TraesCS5A01G153900.5

8.934

0.000

Citrate synthase

TraesCS5B01G416700.1

0.010

0.000

5.358

0.115

Cysteine synthase

TraesCS7A01G102800.1

100.0

0.000

Dehydrin

TraesCS7D01G549900.1

3.395

0.000

Dehydrin

TraesCS6D01G234700.1

0.161

0.000

0.275

0.010

Dehydrin

TraesCS6D01G333000.1

10.38

0.000

0.010

0.000

GST

TraesCS1A01G078800.1

1.325

0.475

6.541

0.019

GST

TraesCS3A01G302100.1

1.510

0.519

100.0

0.000

GST

TraesCS3A01G309000.2

0.010

0.000

1.989

0.779

GST

TraesCS4D01G201100.1

2.141

0.000

1.981

0.779

GST

TraesCS5A01G502300.1

0.898

0.995

9.655

0.000

GST

TraesCS2A01G504300.2

0.010

0.000

1.270

0.979

Peroxidase

TraesCS2D01G107400.1

0.010

0.000

100.0

0.000

Peroxidase

TraesCS7A01G339600.1

1.791

0.095

100.0

0.000

Peroxidase

TraesCS2A01G305600.2

0.450

0.131

100.0

0.000

Peroxidase

TraesCS2A01G263500.1

0.481

0.197

100.0

0.000

Peroxidase

TraesCS5B01G120200.3

1.530

0.772

100.0

0.000

Peroxidase

TraesCS6A01G047200.1

0.847

0.989

100.0

0.000

TraesCS7D01G461500.1

100.0

0.000

100.0

0.000

TraesCS5B01G246900.1

0.010

0.000

2.727

0.621

TraesCS6D01G054800.1

2.438

0.000

2.065

0.887

TraesCS1D01G096400.1

2.440

0.000

1.721

0.932

TraesCS6B01G063800.1

2.793

0.000

2.174

0.654

Peroxidase

TraesCS7A01G319100.1

100.0

0.000

2.635

0.669

Peroxidase

TraesCS2A01G333500.1

0.010

0.000

0.010

0.000

Peroxiredoxin

TraesCS7B01G065200.1

0.492

0.042

2.584

0.464

PP2C

TraesCS2D01G426600.1

100.0

0.000

100.0

0.000

Cu-Zn-SOD

TraesCS7D01G290700.1

1.076

0.922

12.15

0.000

Peroxidase Peroxidase Peroxidase Peroxidase

-p

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Peroxidase

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Gene name

lP

Old leaf

Jo

Young leaf

22

Table 3 Effects of alkali stress on ribosomal protein (RP) abundances in old and young leaves of hexaploid wheat plants. Wheat plants (25 days old) were treated with alkali stress (NaHCO3/Na2CO3, 9:1; 100 mM, pH 8.7) for 4 days. Each treatment and each tissue had four biological replicates, and each biological replicate was a pool of tissues from five plants. Young leaf

Old leaf

Gene ID

Fold change

Adj. P Value

Fold change

Adj. P Value

RP

TraesCS4A01G204800.1

0.010

0.000

0.859

0.761

RP L19

TraesCS2D01G109000.1

0.010

0.000

60S RP L44

TraesCS2B01G246000.1

0.170

0.000

1.870

0.970

60S RP L31

TraesCS6B01G310100.1

0.359

0.000

0.010

0.000

60S RP L22

TraesCS7A01G344500.1

0.413

0.001

1.527

0.979

30S RP S17

TraesCS2B01G623400.1

0.420

0.002

0.426

0.042

30S RP S31

TraesCS7A01G351100.1

0.468

0.006

0.341

0.007

50S RP L27

TraesCS3A01G435700.1

0.465

0.011

0.460

0.096

30S RP S20

TraesCS3A01G268600.1

0.488

0.015

0.397

0.021

50S RP L18

TraesCS4A01G368800.1

0.479

0.016

1.311

0.991

RP L37

TraesCS6A01G386000.1

0.447

0.020

0.669

0.421

50S RP L2

TraesCS4A01G154700.1

0.465

0.025

0.242

0.003

50S RP L15

TraesCS4A01G065400.1

0.494

0.029

0.378

0.021

30S RP S5

TraesCS4D01G151500.4

0.494

0.051

0.253

0.004

50S RP L34

TraesCS3B01G362100.1

0.469

0.059

0.248

0.004

30S RP S6

TraesCS5D01G099800.1

0.297

0.063

0.010

0.000

60S RP l28

TraesCS1A01G363700.1

0.495

0.070

50S RP L20

TraesCSU01G267600.1

0.546

0.150

50S RP L3

TraesCS6A01G105400.1

0.563

0.154

30S RP S17

TraesCS2A01G563100.1

0.558

50S RP L4

TraesCS4A01G091100.1

0.596

30S RP S5

TraesCS4B01G164800.3

0.611

50S RP L11

TraesCS4B01G347000.1

50S RP L21

TraesCS6A01G191400.2

30S RP S16

TraesCS5D01G008200.1

30S RP S9

TraesCS5B01G409400.1

50S RP L2 30S RP S3 30S RP S5 60S RP L18

-p 0.219

0.002

0.428

0.050

re

0.000

0.023

0.267

0.383

0.023

0.330

0.383

0.023

0.643

0.555

0.285

0.001

0.668

0.591

0.381

0.011

0.691

0.685

0.178

0.000

0.700

0.724

0.412

0.022

TraesCS3A01G272200.1

0.718

0.821

0.321

0.004

TraesCS2D01G264000.1

0.740

0.878

0.392

0.022

TraesCS2D01G566300.1

0.760

0.892

0.358

0.014

TraesCS3A01G366100.1

0.783

0.935

0.010

0.000

TraesCS4D01G169400.1

0.809

0.964

0.010

0.000

TraesCS5A01G513500.1

0.807

0.964

0.315

0.006

lP

0.380

Jo

50S RP L5

100.0

0.205

ur na

50S RP L13

ro of

Gene name

50S RP L22

TraesCS2B01G338000.1

0.829

0.976

0.203

0.001

50S RP L23

TraesCS2A01G210900.1

0.950

0.982

0.261

0.002

50S RP L17

TraesCS5A01G468600.2

0.844

0.987

0.323

0.003

50S RP L19

TraesCS6A01G242300.1

0.846

0.989

0.390

0.026

60S RP L7

TraesCS5D01G462800.1

0.934

0.989

0.010

0.000

30S RP S6

TraesCS5D01G530200.1

0.923

0.994

0.353

0.008

30S RP S1

TraesCS4A01G125000.1

0.916

0.994

0.411

0.037

30S RP S6

TraesCS5B01G531900.2

0.899

0.995

0.376

0.013

50S RP L5

TraesCS4D01G339700.1

0.897

0.998

0.414

0.039

23

Table 4 Effects of alkali stress on abundances of ribonucleases and proteinases in old and young leaves of hexaploid wheat plants. 25-days-old-wheat plants were treated with alkali stress (NaHCO3: Na2CO3 = 9:1, 100 mM, pH 8.7) for four days. Each treatment and each tissue had four biological replicates, and each biological replicate was a pool of tissues from five plants. CHFR, E3 ubiquitinprotein ligase CHFR. Young leaf

Old leaf

Gene ID

Fold change

Adj. P Value

Fold change

Adj. P Value

Cysteine proteinase

TraesCS2A01G273100.1

1.747

0.028

7.833

0.000

Cysteine proteinase

TraesCS5A01G326100.1

0.769

0.936

9.262

0.000

Cysteine proteinase

TraesCS2D01G272400.1

0.010

0.000

26.58

0.000

Cysteine protease

TraesCS1B01G338500.1

1.208

0.909

5.116

0.019

Proteasome beta

TraesCS6A01G145800.1

1.648

0.555

100.0

0.000

Ubiquitin

TraesCS4B01G325900.2

0.654

0.841

100.0

0.000

CHFR

TraesCS3D01G079000.2

0.136

0.000

1.642

0.982

Ribonuclease

TraesCS6D01G320200.1

2.551

0.000

5.629

0.004

Ribonuclease

TraesCS6A01G339600.1

3.426

0.000

8.360

0.000

Ribonuclease

TraesCS2B01G182900.1

1.088

0.892

Ribonuclease

TraesCS1A01G152600.1

0.525

0.067

Ribonuclease

TraesCS3D01G392300.1

0.825

0.976

Nuclease S1

TraesCS2D01G507900.1

0.976

re

-p

ro of

Gene name

Jo

ur na

lP

0.994

24

4.332

0.030

0.181

0.004

7.146

0.000

11.32

0.000